Cancer vaccines containing epitopes of oncofetal antigen

ABSTRACT

Disclosed are fragments of oncofetal antigen, otherwise known as immature laminin receptor protein that specifically stimulate one T cell subclass. The fragments may be formulated into compositions for potentiating T cell-mediated responses in mammalian cancer patients. They also have therapeutic uses in vitro.

PRIORITY

This application claims priority on the basis of U.S. provisionalapplication No. 60/400,851, filed Aug. 2, 2002.

STATEMENT REGARDING GOVERNMENTAL SUPPORT

Work leading to the disclosed invention was funded in part by TheNational Institutes of Health grant no. RO1-CA82603-01A251. Therefore,the Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is one of the three leading causes of death in industrializednations. As treatment and preventative measures for infectious diseasesand cardiovascular disease continue to improve, and the average lifeexpectancy increases, cancer is likely to become the most common fataldisease. In developed countries, about one person in three receives adiagnosis of cancer during his or her lifetime and almost one in fourdies from it.

Cancers are the progressive growth of the progeny of a singletransformed cell. A tumor or neoplasm is a population of cells thatexhibit uncontrolled proliferation without regard to normal bodilyrequirements. A malignant neoplasm or cancer is one that threatens lifeby invading and destroying adjacent tissue and/or by seeding(metastasizing) to distant sites. Malignant tumors are divided intocarcinomas (which arise from epithelial precursor cells), sarcomas(which arise largely from mesenchymal tissues) and lymphomas (whicharise from precursors of red and white blood cells). Therefore, curingcancer requires that all the malignant cells be removed or destroyedwithout killing the patient. Unfortunately, the overt manifestation andinitial clinical presentation of cancer usually occur at a late stage inthe disease process when the capacity for invasion has already beenunleashed. By the time of diagnosis, a high proportion of patients haveoccult or even clinically detectable metastases. The capacity ofconventional cytotoxic approaches to succeed in the face of thisadvanced, accelerating disease has, unfortunately, been limited (1,2).In contrast to the short time between disease presentation andestablished metastasis, the period of transition fromhyperproliferative, but noninvasive disease (3-5) to invasive cancer maybe 10 years or more in humans. For breast cancer, this period isestimated to average 6 years (3,4).

A major problem confronting cancer researchers in developingimmunological weapons against this disease is simply that these cellsclosely resemble the normal lineages from which they arise. Thus,despite major advances in the understanding of the factors that lead tothe development of cancer, progress in the clinical management of cancerremains limited. This is due in large part to the limited success ofconventional therapy in the treatment of metastasis. Early researchrevealed that mouse tumors displayed molecules that led to rejection oftumor cells when transplanted into syngeneic (i.e., geneticallyidentical) animals. These molecules are “recognized” by T-cells in therecipient animal, and provoke a cytolytic T-cell response with lysis ofthe transplanted cells. This evidence was first obtained with tumorsinduced by chemical carcinogens. The antigens expressed by the tumorsthat elicited the T-cell response were found to be different for eachtumor. This class of antigens has come to be known as “tumor specifictransplantation antigens” or “TSTAs”. Following the observation of thepresentation of such antigens when induced by chemical carcinogens,similar results were obtained when tumors were induced via ultravioletradiation. See Kripke, J. Natl. Canc. Inst. 53:333-336 (1974).

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytolytic T cells, leadingto tumor cell lysis. This class of immunogenic antigens that arouseT-cell mediated immune reactions in the cancer-bearing host is known as“tumor rejection antigens” or “TRAs”. The extent to which these antigenshave been studied, has been via cytolytic T cell characterizationstudies, in vitro i.e., the study of the identification of the antigenby a particular cytolytic T cell (“CTL” hereafter) subset. The subsetproliferates upon recognition of the presented tumor rejection antigen,and the cells expressing the tumor rejection antigens are lysed.Characterization studies have identified CTL clones that specificallylyse cells expressing the tumor rejection antigens. Examples of thiswork may be found in Levy, et al., Adv. Cancer Res. 24:1-59 (1977);Boon, et al., J. Exp. Med. 152:1184-1193 (1980); Brunner, et al., J.Immunol. 124:1627-1634 (1980); Maryanski, et al., Eur. J. Immunol.124:1627-1634 (1980); Maryanski, et al., Eur. J. Immunol. 12:406-412(1982); Palladino, et al., Canc. Res. 47:5074-5079 (1987).

The immune system responds to cancer cells in complicated ways. Thereare two main types of immune cells that play a significant role incombating disease: B (or bone marrow-derived) lymphocytes (“B cells)produce antibodies to foreign antigens (which constitutes the part ofthe immune system known as humoral immunity); and T (or thymus-derived)lymphocytes (“T cells”) are involved in cell-mediated immunity. Thereare three main subclasses of T cells, namely, helper cells, cytotoxiccells and suppressor cells often referred to as CD4 Th cells, CD8 Tccells and CD8 Ts cells, respectively, on account of their reactivitywith a group (“cluster”) of monoclonal antibodies specific to a surfacemarker that identifies a particular lineage or differentiation stage.Thus, all leukocyte surface antigens whose structures are defined aregiven a “CD” (cluster of differentiation) designation, i.e., CD4 and CD8respectively. The presence of a TRA on a tumor cell is recognized by theT cells and antigen processing cells as a “non-self” or foreign antigen.T cells react with foreign antigens via receptors on their surfaces. Thehuman immune system contains millions of clones of T cells, each ofwhich has distinctive surface receptors. The physical properties ofthese receptors confer specific binding capabilities and permit each ofthe several million clones of T cells in an individual to operateindependently. The T cell receptor is capable of recognizing aparticular antigen only when it is associated with a surface marker onan antigen-presenting cell (APC), such as a dendritic cell or amacrophage. The surface markers belong to a group of molecules known asthe major histocompatibility complex (MHC). Explained in the context ofcancer, a tumor rejection antigen is acquired and processed by APC. TheAPC processes the antigenic protein into shorter peptides calledepitopes that generally range from about 8 to about 12 amino acids inlength. If the peptides are presented on class I MHC proteins to CD8 Tcells, then the epitopes are usually about 8 amino acids in length. Ifthe peptides are presented on class II MHC molecules to CD4 T cells,then the epitopes are usually 9-12 amino acids in length. Binding of theT cell receptor to the epitope of the antigen on the antigen-presentingcell induces changes in the T cell that triggers a cell-mediated immuneresponse.

Two signals are primarily responsible for inducing the T cell mediatedresponse to an APC associated with an epitope of an antigen. A firstsignal results from the binding cross-linking of the T cell receptorswith the epitope:MHC protein complex. A second, co-stimulatory signal issent by “accessory” membrane molecules on the APC when bound by theirreceptors on the responding T cell. Subsequent to the resultingactivation of T cells is the secretion of soluble intercellularmessengers, known generically as “cytokines”, which regulate theamplitude or intensity and duration of the immune response. Cytokinesinclude the group of biomolecules formerly known as lymphokines,monokines, interleukins and interferons (Essential Immunology, seventhedition, Blackwell Scientific Publications, Oxford, Great Britain, 1991,pp. 140-150). In this fashion, T cytotoxic cells that recognize and arespecific to the tumor rejection antigen are stimulated and attack tumorcells that express the antigen.

Malignant tumors have been treated with chemotherapeutic agents thatdirectly impair tumor cells or with immunotherapeutic agents that causenon-specific activation of immunity of a host. In recent years,researchers using tumors of animals, mainly mice, have revealed thattumors can be completely cured by enhancing an antigen-specific immuneresponse to tumor-related antigens and/or tumor-specific antigenspresent in various tumor cells. The treatment has been conducted in theclinic by enhancing the antigen-specific immune response to thesetumor-specific antigens. It is now known, however, that the immuneresponse mediated by the T cells acts either protectively or in asuppressive manner depending upon whether T cytotoxic cells and Tsuppressor cells are activated. Thus, tumor cells can modulateanti-tumor immunity by expressing antigens that preferentially activateTs cells or by secreting cytokines that directly suppress or inducesecretion of suppressive cytokines by T-cells. That is, the activatedCD8 T cells will either recognize and kill the tumor cell carrying theappropriate epitope on its MHC class I molecule, or it will recognizeand become tolerant to the tumor cell, depending on the type of thestimulated CD8 cell, cytotoxic or suppressor, respectively.

Active immunization with some tumor antigens or irradiated, autologoustumor cells themselves has been shown in experimental animals to induceT lymphocyte-mediated immunity which protects the immunized mice fromsubsequent challenge with histocompatible tumor cells (6-8). In variouspreclinical studies (9), immunologic destruction of emerging tumors dueto T lymphocyte recognition of tumor antigens has appeared to involveCD8⁺ cytotoxic T (Tc) cells, but CD4⁺ T helper 1 (Th1) cells have alsobeen shown to be important (10). Within the last few years, a number ofsuch antigens have been identified (8, 11) that appear to be encoded bygenes with tumor-specific expression, expressed in normal cells, butwhich have developed point mutations in the tumor cell, 3) fordifferentiation antigens, or 4) which are over-expressed in certaintumors (12, 13). Many of these tumor antigenic markers will not serve asauto-immunogens when expressed in the host and, therefore, not elicitprotective T lymphocyte responses (11,14). The differentiation antigenswould normally not be expected to raise an immune response due to clonaldeletion of auto reactive T lymphocytes. In some cases, they do becausethe site of normal expression of those genes is in immune-privilegedtissues such as the testis or the eye (11).

The ideal tumor antigen for use in a vaccine or at which to directimmunotherapy would be one which is present on all tumor types, absentor masked in normal tissues, evolutionarily conserved, and its functionrequired for the malignancy of the tumor cells. Such an immunogen wouldbe less likely to be able to be down regulated or mutated and still havethe tumor cells grow and metastasize optimally. Thus, if tumor cellsused such mechanisms to evade the immune response to that immunogen(15), the tumor cells would be reducing their ability to thrive.

Applicants discovered that tumor cells express a common antigen whichwas originally called oncofetal antigen (OFA). This protein was detectedin early to mid gestation fetal cells, hence the term “OncofetalAntigen”. It is comprised of a single polypeptide chain of 295 aminoacids and has a molecular weight of about 37-44 kDa. OFA was identifiedby Applicants to be a universal tumor specific transplantation antigenas it was detected on chemical or irradiation induced rodent tumors. Alltumors that Applicants have tested were shown to express OFA (1, 43,44). The tumors include chemically- and virally-induced sarcomas,X-irradiation-induced T cell lymphomas, and many tumors of inbredrodents reported by others to express only a unique, non-shared TSTA.Besides rodent tumors, approximately 500 human tumors representing mostcancer types have been tested—all were found to express OFA (43-45). Forexample, OFA is also expressed by carcinomas of the breast, kidney,lung, colon, gastric mucosa, larynx, pharynx, ovary and prostate whereasnormal tissues of the same types do not express OFA (43-45). OFA isbelieved to play an important role in tumor progression and has beenimplicated in tumor invasiveness, metastasis and growth.

Oncofetal antigen has recently been cloned. Complementary DNA sequencealignments have revealed 99% identity with another human protein calledimmature laminin receptor protein (iLRP). Hence, these two proteins arebelieved to be identical. (Hereinafter, the terms “OFA,” “iLRP,”“OFA/iLRP” and “iLRP/OFA” are used interchangeably.) The mature form ofthis laminin receptor appears to be a dimer of acylated immature 32 kDalaminin receptor protein (iLRP) (16). Although the mature 67 kDa form ison many normal cells as well as on tumor cells, there appears to be apreferential expression of the 32 kDa iLRP by fetal and tumor cells (17,18). The iLRP is evolutionarily conserved (19). Indeed, the amino acidsequence of the human iLRP differs from that of murine iLRP by only fouramino acids (20).

Tumor invasion of host tissues and trophoblastic penetration of theendometrium share common biological features. Both processes involve theinvasion of basement membrane, an event that is initiated by adhesion ofcancer or trophoblast cells to basement membrane components andparticularly to laminin. Adhesion to laminin is mediated through avariety of cell surface receptors. Other investigators (Van den Brule FA, et al., Biochem. Biophys. Res. Commun. 201:388-393 (1994)), haveshown that the 67 kD laminin receptor (67LR) and galectin-3 areinversely modulated as the invasive phenotype of cancer cellsprogresses, with up regulation of the former, and down regulation of thelatter, respectively. These investigators found that the 67LR expressionlevels in the fetus increased from the 7^(th) week of gestation to amaximum at the 12^(th) week, when invasion is maximal, and thendeclined. Expression of galectin-3 was inversely modulated by thegestational age, with a minimum expression at the 12^(th) week ofgestation. A year earlier (1993), and 6 years before our identificationof Oncofetal Antigen as iLRP, Applicants reported (in Coggin et al.,Arch. Otolaryngol. Head Neck Surg. 119:1257-1266 (1993)) that based onthe results of flow cytometry using different strains of mice, that theproportion of cells expressing OFA increased gradually during thegestational life of the fetus to reach its maximum levels (29% of thecells) at mid-gestation (day 13) and thereafter dropped gradually to 5%at day 18, whereas newborn mice did not show increased levels ofexpression of OFA.

The transition from in situ tumor growth to metastatic disease isdefined by the ability of tumor cells of the primary site to invadelocal tissues and to cross tissue barriers. To initiate the metastaticprocess, cancer cells must adhere to extracellular matrix (ECM)components, secrete proteases which digest the dense matrix of type IVcollagen, glycoproteins, and proteoglycans allowing them to invade theinterstitial stroma and respond to factors inducing motility of theinvasive cells (21). For distant metastases, intravasation requirestumor cell invasion of the subendothelial basement membrane of bloodvessels using the same mechanisms (22). Several published experimentshave suggested that tumor cell interaction with the laminin component ofthe ECM is important to the expression of the metastatic phenotype (23,24). Upon binding of laminin by the immature form of the high affinitylaminin receptor (iLRP), its expression and that of the laminin-bindingα6β1 or 4 integrin are enhanced (25, 26). Thus, the stability of lamininbinding by the tumor cells is enhanced. Besides this, the same stepinduces production and secretion of the collagenase IV matrixmetalloproteinases (27, 28) required for digestion of the ECM to allowmetastasis to occur. Increased expression of collagenase IV is seen ininvasive colonic, gastric, ovarian, and thyroid adenocarcinomas whilebenign proliferative disorders of the breast and colon and normalcolorectal and gastric mucosa have low or no staining for theseproteases (29,30). Increased expression of iLRP is also seen in a widevariety of human adenocarcinomas, including those of the colon, breast,stomach, and liver (29, 31). Over-expression of iLRP is associated withpoor prognosis in several types of tumor (32-35). In breast carcinoma,over-expression of iLRP correlates with early dissemination of the tumorcells to the bone marrow that further emphasizes the role of iLRP in themetastatic process (36). Experimental administration of anti-iLRPantibody or anti-laminin antibody at the time of tumor cell injectioninhibits tumor metastasis (37-39).

OFA/iLRP is immunogenic. OFA/iLRP-specific T cells cloned fromirradiated mice have been identified as Th1-type CD4+ T cells, whichproduce interferon-gamma, or cytotoxic T cells which secreteinterferon-γ. Also, CD8+ suppressor T cells, which secrete IL-10 areinduced. In addition, stimulating peripheral blood mononuclear cellsfrom patients with breast cancer with autologous tumor cells resulted inthe expansion of tumor reactive T cells. Analysis of these tumorreactive T-cells cloned by Applicants revealed that a substantialproportion of the clones showed reactivity against purified OFA/iLRP.

In more recent experiments, Applicants have observed that immunizationof mice with syngeneic tumor cells expressing iLRP resulted incross-reactive protective immunity against a spectrum of syngeneictumors because they all express iLRP (6, 7). Immunization withiLRP:nitrocellulose particles produced distinct T and B cell mediatedimmunity depending on the dose of iLRP used. Thus, immunization with theintact iLRP protein can induce effector or regulatory T cells dependingon the dose used.

The OFA/iLRP also activates T_(s) cells. They secrete IL-10. T_(s) cellsprevent T_(c) cells from exhibiting cytotoxic activity against tumorcells. Once the concentration of iLRP reaches a certain optimalconcentration, it induces IL-10-producing Ts cells that prevent T_(c)cells from killing antigen-positive target tumor cells. This phenomenoncaused by an excess of the T cell immunogen, 37-44 kDaOFA, enables theimmune system to suppress T_(c)-mediated immunity. In other words, it isan immuno-regulatory controlled measure that prevents over-production ofT_(c) cells to any T_(c)-antigen. This immuno-regulation preventsanti-self T_(c)-mediated immunity and other anti-self immunity.

Rohrer et al. (40) showed that the apparent tumor-free, long-termsurvivors of fractionated, sublethal x-irradiation had developediLRP-specific memory Th1 and Tc lymphocytes even though they showed nosign of lymphoma development. Approximately, half of the RFM mice thatwere irradiated died within 6 months after irradiation with metastaticthymic lymphoma (41). Besides the memory effector Th1 and Tc lymphocytesinduced by iLRP during tumor development, non-cytotoxic, iLRP-specific,CD8⁺ T cells that secreted IL-10 upon antigen stimulation were alsocloned from those long-term RFM mouse radiation survivors (40,42). TheIL-10 inhibited Tc activity (42) and so these cells can dampenanti-tumor immunity of whatever specificity. We suggested that the timeof appearance and/or the relative number of IL-10-secreting CD8 Tlymphocytes compared to that of iLRP-specific Tc cells may have been afactor in determining whether an irradiated RFM mouse developed a thymiclymphoma and died from it subsequent to X-irradiation (43). In thisregard, Applicants have observed that during breast or renal cellcarcinoma development in humans, iLRP-specific Th1, Tc, andIL-10-secreting, CD8⁺ T (Ts) lymphocytes were clonable from thepatients' peripheral blood (44, 45). Consistent with their view of thecontribution of the Ts cells to tumor progression (43), Applicants havealso found that breast cancer patients with the highest ratio ofiLRP-specific Ts:Tc lymphocytes required a second surgery due to tumorrecurrence (44). Thus, the frequency of the IL-10-secreting,iLRP-specific Ts lymphocytes in cancer patients may be used as aprognostic for clinical response to therapy (44). Such methods are asubject of U.S. Pat. No. 6,335,174.

Thus, while use of OFA/iLRP for cancer therapy and as a vaccine holdspromise, it is tempered by the possibility that such uses will alsotrigger Ts-mediated immuno-regulation. In this regard, Rohrer et al.,Mod. Asp. Immunobiol. 1(5):191-195 (2001), state that it is important todefine the peptide epitopes which stimulate iLRP/OFA-specific Tc, Th andthe IL-10 secreting Ts cells in order to determine if the epitopes whichstimulate the Ts cells are different than and located on a differentportion of the OFA protein than the epitopes that stimulate the Tcand/or Th cells.

SUMMARY OF THE INVENTION

The speculation in the Rohrer publication aside, the facts remain thatOFA/iLRP-specific Tc and Ts cells are both CD8 T cells and that with theexception of the spectrum of cytokines that they produce, theirfunctionally abilities are basically the same. Applicants had also shownthat Ts cells display Tc-like cytotoxic activity in the presence ofanti-IL-10 antibodies (which neutralize the IL-10 secreted by the Tscells). Further, the Rohrer publication also demonstrated that therelative stimulation of Tc and Ts cells by OFA/iLRP in mice wasdose-dependent; since Ts cells have lower affinity T cell antigenreceptors (TCRs) compared to TCRs on Tc cells, Tc cells responded tosignificantly smaller doses of OFA/iLRP than Ts cells. These findingssuggested that dosage amount (as opposed to the epitope itself) is animportant variable in potentiating an immune response withoutstimulating Ts cells. On the basis of these facts and observations,persons skilled in the art would have expected Tc and Ts cells to bereactive to the same spectrum of OFA epitopes.

Applicants have now discovered distinct, non-overlapping OFA fragmentscontaining epitopes that stimulate one class or subclass of T cellsversus other classes. One aspect of the present invention is directed toOFA epitopes that specifically stimulate Tc cells. Another aspect of thepresent invention is directed to OFA epitopes that specificallystimulate Ts cells. Yet another aspect of the present invention isdirected to OFA epitopes that specifically stimulate Th cells. DNAsencoding the OFA fragments and epitopes, and methods of making theepitopes are also provided.

Another aspect of the present invention is directed to a method foridentifying epitopes of mammalian OFA that stimulate T cytotoxic cellsor T suppressor cells relative to other T cells, in mammals. The methodentails a) obtaining a sample of peripheral blood leukocytes (PBLs) orsplenocytes from a tumor-bearing mammal; b) clonally expanding T cellsof different T cell subclasses present in the sample and that arespecific to OFA, thus producing clones of T cells of different T cellsubclasses; c) determining subclass type of each of the clones of T cellsubclasses; d) culturing the clones of T cells of (c) in the presence ofa deletion mutant of OFA; and e) comparing extent of stimulation ofclones of T cells of one subclass by the OFA deletion mutant tostimulation of clones of T cells of other T cell subclasses by the OFAdeletion mutant; wherein greater stimulation of a clone of T cells ofone subclass relative to that of other T cell subclasses by the OFAdeletion mutant is indicative that the OFA deletion mutant contains anepitope that stimulates T cells of one subclass relative to the other Tcell subclasses.

In some embodiments, the sample contains splenocytes obtained from amouse. In other embodiments, the mammal is a human and the samplecomprises PBLs. In some embodiments, (e) comprises comparing stimulationof the clones of T cells of the subclass by the OFA epitope to twocontrols, wherein the first control comprises intact OFA and the secondcontrol comprises an OFA mutant that lacks the epitope. In yet otherembodiments, (d) and (e) are repeated using a plurality of OFA deletionmutants wherein each OFA deletion mutant lacks a different portion ofthe entire OFA molecule.

OFA epitopes disclosed herein are therapeutically useful in mammals.Accordingly, a further aspect of the present invention is directed tocompositions containing at least one OFA epitope that stimulates orinduces T cytotoxic cells. In preferred embodiments, this aspect of thepresent invention is directed to an immunotherapeutic composition e.g.,a vaccine, comprising or consisting essentially of a plurality of OFAepitopes that specifically stimulate T cytotoxic cells, and a carrier(and in preferred embodiments, a carrier which also functions as animmunopotentiating adjuvant). By the phrase “consisting essentially of”it is meant to exclude elements that would affect the basic and novelcharacteristics of the composition such as its immunogenic effect interms of stimulating T cytotoxic cells relative to T suppressor cells.Thus, elements that would be excluded from the compositions include OFAepitopes (or regions of the OFA protein that contain such epitopes) thatspecifically stimulate T suppressor cells because their presence wouldcause a diminution of the therapeutic effect of the composition. Thecompositions may also include one or more OFA epitopes that specificallystimulate Th cells. The compositions stimulate proliferation ofOFA-specific Tc cells, thus potentiating T cell-mediated immunity inmammalian cancer patients, which may inhibit growth or proliferation ofcancer cells and/or induce immunity.

A further aspect of the present invention is directed to methods ofmaking a vaccine or immunotherapeutic composition. The method entails(a) identifying a plurality of oncofetal antigen (OFA) epitopes thatspecifically stimulate T cytotoxic lymphocytes in the mammal (e.g.,human); and (b) formulating two or more of the epitopes identified in(a) with a carrier, thus forming the immunotherapeutic composition. Inother embodiments, the method further entails c) identifying a pluralityof oncofetal antigen (OFA) epitopes that specifically stimulate T helperlymphocytes in the mammal (e.g., human), and wherein b) comprisesformulating one or more of the OFA epitopes identified in c) with thetwo or more epitopes identified in a), along with the carrier.

The OFA epitopes of the present invention that specifically stimulate Tccells and optionally, the OFA epitopes that specifically stimulate Thcells may be administered to cancer patients, preferably together in theform of a composition. Thus, the present invention further provides amethod of treating cancer in a mammal, by administering to a cancerpatient at least one and preferably a plurality of oncofetal antigen(OFA) epitopes that specifically stimulate T cytotoxic lymphocytes inthe mammal, and optionally, one and preferably a plurality of oncofetalantigen (OFA) epitopes that specifically stimulate T helper lymphocytesin the mammal. A related aspect of the invention is directed to a methodof potentiating a T cell-mediated immune response in a mammalian cancerpatient comprising administering to the cancer patient an immunogenicamount of a composition as described herein. The OFA epitopes of thepresent invention provide a refined, customized approach for selectivelystimulating the immune system to enhance cytotoxic anti-tumor cellactivity. Embodiments of the present invention serve to reduceimmuno-regulation associated with the stimulation of T suppressor cellscaused by intact OFA/iLRP, thus allowing T cytotoxic cells to continuemounting an attack against tumor cells, especially when tumor burdenincreases.

DETAILED DESCRIPTION OF THE INVENTION

Below is an alignment of the full-length cDNA sequence of 37 kDa OFAfrom MCA-1315 murine fibrosarcoma with the nucleotide sequence of themurine iLRP (reported in Rao, et al., Biochemistry 28:7476-7480 (1989)).The predicted amino acid is indicated under the nucleotide sequence. Thestop codon is indicated by an asterisk. Nucleotide sequences in lowercase print preceding the 5′ end and following the 3′ end are for SalIand NotI sites respectively, that were used in cloning. The amino acidsequences of the two peptides (a.a. residues 18-40 and 43-52) that werederived from mAb115-affinity purified OFA are underlined. The sequencealignment revealed 99.5% identity between the nucleotide sequence of OFAand published nucleotide sequence for murine iLRP. The predicted aminoacid sequence of OFA and the amino acid sequence encoded by mouse iLRPgene were 99.3% identical. The only differences were amino acid residues18 and 155 which were phenylalanine and arginine for OFA instead ofleucine and alanine in the published amino acid sequence of murine iLRP,respectively, but which were identical to those of the human iLRPsequence. (SEQ ID NOS 1 & 3 are the DNA sequences and SEQ ID NOS 2 & 4are the encoded proteins, respectively)

As shown below, the murine OFA and murine iLRP share 99.3% sequencesimilarity; there are only two differences in amino acids in the entire295 amino acid sequence. Likewise, mouse OFA and human iLRP differ in 2amino acids in their sequences. See, Rao, et al., Biochemistry28:7476-7486 (1989) (murine iLRP); Yow, et al., PNAS 85:6394-6398 (1988)(human iLRP); and Coggin, et al., Anticancer Res. 19:5535-5542 (1999)(murine OFA). (SEQ ID NOS 4, 5 & 2, respectively, in order ofappearance)

Mu iLRP M S G A L D V L Q M K E E D V L K L L A 20 HuiLRP - - - - - - - - - - - - - - - - - F - - MuOFA - - - - - - - - - - - - - - - - - F - - A G T H L G G T N L D F Q ME Q Y I Y K 40 Hu iLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - R K S D G I Y I I N L K R TW E K L L L 60 Hu iLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - A A R A I V A I E N P A D VS V I S S R 80 Hu iLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - Mu iLRP N T G Q R A V L K FA A A T G A T P I A 100 Hu iLRP - - - - - - - - - - - - - - - - - - - -Mu OFA - - - - - - - - - - - - - - - - - - - - Mu iLRP G R F T P G T F TN Q I Q A A F R E P R 120 HuiLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - Mu iLRP L L V V T D P R A DH Q P L T E A S Y V 140 Hu iLRP - - - - - - - - - - - - - - - - - - - -Mu OFA - - - - - - - - - - - - - - - - - - - - Mu iLRP N L P T I A L C NT D S P L A Y V D I A 160 Hu iLRP - - - - - - - - - - - - - -R - - - - - Mu OFA - - - - - - - - - - - - - - R - - - - - Mu iLRP I P CN N K G A H S V G L M W W M L A R 180 HuiLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - Mu iLRP E V L R M R G T I SR E H P W E V M P D 200 Hu iLRP - - - - - - - - - - - - - - - - - - - -Mu OFA - - - - - - - - - - - - - - - - - - - - Mu iLRP L Y F Y R D P E EI E K E E Q A A A E K 220 HuiLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - Mu iLRP A V T K E E F Q G EW T A P A P E F T A 240 Hu iLRP - - - - - - - - - - - - - - - - - - - -Mu OFA - - - - - - - - - - - - - - - - - - - - Mu iLRP A Q P E V A D W SE G V Q V P S V P I Q 260 Hu iLRPT - - - - - - - - - - - - - - - - - - - Mu OFAA - - - - - - - - - - - - - - - - - - - Mu iLRP Q F P T E D W S A Q P AT E D W S A A P 280 Hu iLRP - - - - - - - - - - - - - - - - - - - - MuOFA - - - - - - - - - - - - - - - - - - - - Mu iLRP T A Q A T E W V G AT T E W S 295 Hu iLRP - - - - - - - - - - - - D - - MuOFA - - - - - - - - - - - - E - - Amino Acid Abbreviations: Alanine AArginine R Asparagine M Aspartic Acid D Cysteine C Glutamine Q GlutamicAcid E Glycine G Histidine H Isoleucine I Leucine L Lysine K MethionineM Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine YValine V

Thus, for purposes of the present invention, murine OFA, murine iLRP,human OFA and human iLRP are collectively referred to as “OFA”, and asindicated above, “OFA” and “iLRP” are used interchangeably along with“OFA/iLRP” and “iLRP/OFA”. By “OFA,” it is intended to mean a consensus295 amino acid polypeptide with variability in positions 18, 155, 241and 293 as shown. Any epitope containing an amino residue that is notcommon to all the aforementioned OFA and iLRP proteins as shown above,or any other mammalian OFA or iLRP, may be considered to contain at theleast, variability in that position.

One aspect of the present invention is directed to epitopes of OFA thatstimulate proliferation of T cells belonging to one subclass relative toone or more other subclasses; that is, they specifically stimulate Tc,Th or Ts cells. Relative or specific stimulation may be compared to acontrol such as IMDM (Iscove's Modified Dulbecco's Medium). Statedsomewhat differently, the stimulation of a given subclass of T cells bythe OFA epitope will be comparable to if not greater than the amount ofstimulation of the given subclass of T cells by intact OFA. Relativestimulation of subclasses of murine T cells is quantified in theexamples below. As shown in various tables in the examples below, OFAepitopes that stimulate Tc cells show as much as a 56-fold increase instimulation of Tc cells versus Ts cells. OFA epitopes that stimulate Tscells show as much as a 13-14-fold increase in stimulation of Ts cellsversus Tc cells. Thus, unlike intact OFA, the stimulation of the other Tcell subclasses induced by the epitope is comparable to a baseline orcontrol (e.g., 2-10-fold difference with a control such as IMDM, asshown in Table 4 below). Thus, in general, by the phrase “an OFA epitopethat specifically stimulates one T cell subclass (Tc, Ts or Th),” it ismeant that the stimulation of that given subclass of T cells is at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60-fold, or more,compared to at least one of the other T cell subclasses, particularly asbetween Tc and Ts cells, and Ts and Th cells.

The OFA epitopes of the present invention typically have a length offrom 8-12 amino acids, although slightly shorter or longer peptides maybe used, provided that they also exhibit substantially the sameproperties in terms of stimulation of T cells. Thus, the epitopes mayalso be described in terms of a peptide of from about 8 or 8 to about 12or 12 amino acids. In the case of Th cells, epitopes can be longer e.g.,up to about 20 or about 25 amino acids in length, due to differencesbetween class I and class II MHC protein binding and presentingantigenic peptides.

OFA epitopes that specifically stimulate proliferation of clones of Tccells (relative to Ts and Th) cells at least in mice have been found inthe N-terminal and C-terminal regions of OFA. Tc epitopes containingamino acid residues 53-60 (e.g., RTWEKLLL) (SEQ ID NO: 6) or residues81-88 (e.g., NTGQRAVL) (SEQ ID NO: 7), have been identified in theN-terminal region, and Tc epitopes containing residues 229-236(GEWTAPAP) (SEQ ID NO: 8) have been identified in the C-terminal region.Thus, other Tc epitopes embraced by the present invention include OFA(49-60), OFA (50-61), OFA (51-62), OFA (52-63), OFA (53-64), OFA(50-60), OFA (51-61), OFA (52-62), OFA (53-63), OFA (51-60), OFA(52-61), OFA (53-62), OFA (52-60), OFA (53-61), OFA (77-88), OFA(78-89), OFA (79-90), OFA (80-91), OFA (81-92), OFA (78-88), OFA(79-89), OFA (80-90), OFA (81-91), OFA (79-88), OFA (80-89), OFA(81-90), OFA (80-88), OFA (81-89), OFA (225-236), OFA (226-237), OFA(227-238), OFA (228-239), OFA (229-240), OFA (226-236), OFA (227-237),OFA (228-238), OFA (229-239), OFA (227-236), OFA (228-237), OFA(229-238), OFA (228-236) and OFA (229-237). Other preferred epitopesinclude sequences of about 8-12 amino acids and which contain CNTDSPLR(SEQ ID NO: 9) (e.g., amino acid residues 148-155) or the sequenceYVDIAIPC (SEQ ID NO: 10)(e.g., amino acid residues 156-163). Thus, asidefrom these two sequences, additional Tc epitopes include OFA (144-155),OFA (145-156), OFA (146-157), OFA (147-158), OFA (148-159), OFA(145-155), OFA (146-156), OFA (147-157), OFA (148-158), OFA (146-155),OFA (147-156), OFA (148-157), OFA (147-155), OFA (148-156), OFA(152-163), OFA (153-164), OFA (154-165), OFA (155-166), OFA (156-167),OFA (153-163), OFA (154-164), OFA (155-165), OFA (156-166), OFA(154-163), OFA (155-164), OFA (156-165), OFA (155-163), and OFA(156-164). As per the alignment shown above, any epitopes thatcorrespond to a fragment of OFA containing residue 155 could contain an“A” residue in its place. Other preferred epitopes include the sequenceTIALCNTDS (SEQ ID NO: 11) (e.g., amino acid residues 144-152), TDSPLRYVD(SEQ ID NO: 12) (e.g., amino acid residues 150-158), PLRYVDIAI (SEQ IDNO: 13)(e.g., amino acid residues 153-161, and VDIAIPCNN (SEQ ID NO:14)(e.g., amino acid residues 157-165). There may be in certain fewinstances, epitopes that are recognized by both Ts and Tc cells.However, due to the lower affinity of antigen receptors on Ts cells,when OFA/iLRP is present in limited amounts, the Tc cells will bepreferentially stimulated or induced. The antigen receptors on Tc cellshave relatively higher affinity for OFA/iLRP peptide:MHC proteincomplex, in order to give an activation signal to the Tc cell.

OFA epitopes that specifically stimulate Th cells relative to Tc and Tscells at least in mice have been identified to include the sequenceSPLRYVDIAI (SEQ ID NO: 15) (e.g., amino acid residues 152-161 of OFA).Additional OFA epitopes that specifically stimulate Th cells relative toTc and Ts cells at least in mice have been found in the C-terminalregion of OFA. Preferred epitopes in this region contain the sequencesOFA (229-238) (e.g., GEWTAPAPEF) (SEQ ID NO: 16), OFA (241-250) (e.g.,AQPEVADWSE) (SEQ ID NO: 17), OFA (253-262) (e.g., QVPSVPIQQF) (SEQ IDNO: 18), OFA (277-286) (e.g., SAAPTAQATE) (SEQ ID NO: 19) and OFA(285-294)(e.g., TEWVGATTDW) (SEQ ID NO: 20). Thus, OFA epitopes thatspecifically stimulate Th cells may contain one of these sequences, andbut may also contain additional amino acids on either or both termini,to reach lengths of about 20 to about 25 amino acids. On the other hand,they may contain about 8 or 9 of the 10 amino acids (e.g., OFA (152-159,OFA (153-160), OFA (154-161), OFA (152-160), OFA 153-161), OFA(229-236), OFA (230-237), OFA (231-238), OFA (229-237), OFA (230-238),OFA (241-248), OFA (242-249), OFA (243-250), OFA (241-249), OFA(242-250), OFA (253-260), OFA (254-261), OFA (255-262), OFA (253-261),OFA (254-262), OFA (277-284), OFA (278-285), OFA (279-286), OFA(277-285), OFA (278-286), OFA (285-292), OFA (286-293), OFA (287-294),OFA (285-293 and OFA (286-294).

OFA epitopes that specifically stimulate proliferation of Ts cells(relative to Tc and Th cells) at least in mice have been found in theregion containing amino acid residues 9-28 and which contain thetetrapeptide KLLA (SEQ ID NO: 21) (e.g., amino acid residues 17-20), andpreferably the octapeptide KLLAATGH (SEQ ID NO: 22) (e.g., amino acidresidues 17-24). Thus, representative OFA epitopes include OFA (9-20),OFA (10-20), OFA (11-20), OFA (12-20), OFA (13-20), OFA (10-21), OFA(11-21), OFA (12-21), OFA (13-21), OFA (14-21), OFA (11-22), OFA(12-22), OFA (13-22), OFA (14-22), OFA (15-22), OFA (12-23), OFA(13-23), OFA (14-23), OFA (15-23), OFA (16-23), OFA (13-24), OFA(14-24), OFA (15-24), OFA (16-24), OFA (17-24), OFA (14-25), OFA(14-25), OFA (15-25), OFA (16-25), OFA (17-25), OFA (15-26), OFA(16-26), OFA (17-26), OFA (16-27), OFA (17-27) and OFA (17-28).

Additional OFA epitopes that specifically stimulate proliferation ofclones of Ts cells (relative to Tc and Th cells) at least in micecontain amino acid residues 37-44 (e.g., IYKRKSD) (SEQ ID NO: 23) andresidues 97-104 of OFA (e.g., TPIAGRFT) (SEQ ID NO: 24). Thus, asidefrom these two sequences, additional Ts epitopes include OFA (33-44),OFA (34-45), OFA (35-46), OFA (36-47), OFA (37-48), OFA (34-44), OFA(35-45), OFA (36-46), OFA (37-47), OFA (35-44), OFA (36-45), OFA(37-46), OFA (36-44), OFA (37-45), OFA (93-104), OFA (94-105), OFA(95-106), OFA (96-107), OFA (97-108), OFA (94-104), OFA (95-105), OFA(96-106), OFA (97-107), OFA (95-104), OFA (96-105), OFA (97-105), OFA(96-104) and OFA (97-105). Yet other OFA epitopes that stimulateproliferation of clones of Ts cells (relative to Tc and Th cells) atleast in mice contains the sequence VNLPTIAL (SEQ ID NO: 25) (e.g., OFA(140-147). Thus, aside from OFA (140-147), a further list ofrepresentative OFA epitopes that stimulate Ts cells includes OFA(136-147), OFA (137-148), OFA (138-149), OFA (139-150), OFA (140-151),OFA (137-147), OFA (138-148), OFA (139-149), OFA (140-150), OFA(138-147), OFA (139-148), OFA (138-146, OFA (138-147), OFA (138-148),OFA (138-149), OFA (140-149), OFA (139-147) and OFA (140-148).

The above-referenced publication by Rohrer et al., in Modern Aspects ofImmunobiology further states that the task of identifying OFA epitopesthat potentiate anti-OFA/iLRP anti-tumor mediated immunity will probablynot be that simple, especially when the human outbred MHC is taken intoconsideration. Despite this consideration, as well as the differencesbetween the major histocompatibility class antigens in humans and theirH2 counterparts in mice, Applicants have now come to believe that OFAepitopes disclosed herein are also functional in humans. See the examplebelow entitled “Conformation of OFA Epitope binding to H-2^(d) Class IProteins.” Regardless, OFA epitopes functional in a given mammal such asa human may be identified or confirmed in accordance with the methoddescribed below. To identify (or as the case may be, to confirm theidentity of) epitopes of OFA that selectively stimulate proliferation ofone subset of T cells versus one or more other subsets of T cells in agiven mammal such as a human, a sample of peripheral blood mononuclearleukocytes (PBMLs) (or mononuclear leukocytes (MNLs) derived from spleenor lymph nodes) are obtained from a tumor-bearing mammal. Since OFA hasbeen found to be a universal tumor rejection antigen in all themalignant systems tested to date, the method may be practiced with PBMLsor MNLs from any tumor-bearing mammal, including humans. The sample isthen cultured in a medium containing a predetermined concentration ofOFA and one or more growth factors required for growth of T cells (e.g.,IL-2 and IL-6) and antigen processing cells (APCs), so as to allowexpansion of the T cells present in the sample. APCs are typicallypresent in a PBML or MNL sample. Thus, when initially establishing thetumor-reactive lymphocytes in culture, no additional APCs need to beadded. However, in order to be able to restimulate and clone thosereactive T lymphocytes subsequently, additional irradiated autologous(human) or syngeneic (mouse) APCs are added along with the OFA epitopeor deletion mutant used for stimulation. As a result of this procedure,clones of T cells that recognize OFA may be identified. They are thencounted, followed by dilution and plating out. Preferably, the limiteddilution analysis entails plating the T cells out into wells to achievea Poisson-Type distribution (e.g., wherein after terminal dilution,greater than about 37% of the wells “plated” with test lymphocytes willhave no reactive T lymphocytes and dilutions are made such that there isa 90% probability that any T lymphocyte colonies that form each camefrom only one cell and, thus may be properly considered as clones.Following the plating out e.g., into plastic microwells, APCs, OFA andgrowth factors are added to each well. This procedure results in theproduction of clones of T cells that are specific to OFA.

Following the cloning procedure, the T cell clones are identifiedaccording to subclass type. This procedure may be accomplished inaccordance with standard techniques. For example, helper T cells may bedistinguished from both T suppressor and T cytotoxic cells bydetermining their reactivity with anti-CD4 and anti-CD8 antibodies. Thcells react with anti-CD4 antibodies and T_(s) and T_(c) both react withanti-CD8 antibodies. Reactivity with such antibodies may be determinedin accordance with standard techniques such as flow cytometry. Todistinguish T_(s) verses T_(c) cells, the culture medium is analyzed todetect presence of IL-10. This interleukin is produced by T_(s) but notT_(c) cells. Although the Tc cells may be identified by default, apositive determination can also be made by analyzing the culture mediumfor the presence of the cytokine IFN-gamma which these cells (Tc) make(but which T_(s) cells do not make) and an in vitro cytotoxicity test(i.e., demonstrating that these cells kill tumor cells) may also beconducted to confirm the presence of T_(c).

Once the subsets of T cells specific to OFA have been cloned andidentified, they are cultured once again with the same aforementionedingredients except that on this occasion, a truncated OFA proteinproduced by an OFA deletion mutant is added to the medium. By “OFAdeletion mutant” it is meant any segment of the 295 amino acid sequenceof OFA. For example, the deletion mutant may constitute a fragment ofOFA (e.g., amino acids 1-25 or 250-295) or the intact 295 amino acidsOFA less a deletion of internal amino acids (e.g., OFA mutant containingamino acid residues 1-135 and 156-295). The relative stimulation of Th,Ts or Tc cells by the OFA deletion mutant protein may be determinedusing standard procedures as well. The extent of stimulation of theclones may be determined, for example, by measuring uptake by the cellsof a detectably labeled nucleotide in the culture medium, such as³H-thymidine or by ELISA detection of 5-bromodeoxyuridine (BudR)incorporation. In addition, the determination may be made using positiveor negative tests. A “positive-type” test simply entails a comparison ofthe relative stimulation of the T cell clones to a single OFA deletionmutant protein added to the culture. In preferred embodiments, the testis done in a negative “manner”, which uses a plurality of overlappingOFA deletion mutants, wherein the deletions taken collectivelycorrespond to the entire OFA protein. In either case, it is preferred tocompare the determined value for any given T cell clone against acontrol such as intact OFA per se. If the method is initially carriedout with OFA deletion mutant proteins greater than about 12 amino acidsin length, the stimulation of the T cell clones in subsequentdetermination of relative stimulation should be conducted at least oneadditional time, each time using a shorter OFA deletion mutant in orderto identify an OFA epitope that produces maximum relative stimulation ofT cells of a given subclass relative to the others.

This method may also be used to test analogs of the epitopes, e.g., thatdiffer from the naturally occurring sequence in terms of one or morenaturally or non-naturally occurring amino acid substitutions oradditions, or one or more amino acid deletions. As stated above, thismethod may be used to determine whether epitopes corresponding tosequences containing amino acid positions 18, 155, 241 and/or 293 maycontain the amino acid residue native to human or murine iLRP.Modifications and changes may be made in the structure of the OFAepitope provided that the modification or change does not alter theepitope to the point where it does not selectively stimulate the givensubclass of T cells. Such are termed “biologically functionalequivalents,” “functional equivalents” or “analogs,” are alsoencompassed within the meaning of the term “OFA epitope”.

For example, one of skill in the art will recognize that certain aminoacids may be substituted for other amino acids in a given OFA epitope.It is also well understood by the skilled artisan that there is a limitto the number of changes that may be made within a portion of themolecule and still result in a molecule with an acceptable level ofequivalent biological activity. In determining whether a givensubstitution, addition or deletion will result in a significant changein the desired activity, there are several general guidelines toconsider. In particular, where shorter length epitopes are concerned, itis contemplated that fewer amino acids should be made within the givenpeptide. Longer epitopes may have an intermediate number of changes. Thelongest epitopes will have the most tolerance for a larger number ofchanges. It is also well understood that where certain residues areshown to be particularly important to the biological or structuralproperties of a polyamino acid, such residues may not generally beexchanged. Amino acid substitutions are generally based on the relativesimilarity of the 5 amino acid side-chain substituents, for example,their hydrophobicity, hydrophilicity, charge, size, and the like. Ananalysis of the size, shape and type of the amino acid side-chainsubstituents reveals that arginine, lysine and histidine are allpositively charged residues; that alanine, glycine and serine are all asimilar size; and that phenylalanine, tryptophan and tyrosine all have agenerally similar shape.

Therefore, based upon these considerations, members of the followinggroups, namely: arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine are defined herein asbiologically functional equivalents. To effect more quantitativechanges, the hydropathic index of amino acids may be considered. Eachamino acid has been assigned a hydropathic index on the basis of itshydrophobicity and charge characteristics, which are as follows:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein, and correspondingly apolyamino acid, is generally understood in the art. It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within 2 ispreferred, those which are within approximately 1 are particularlypreferred, and those within approximately 0.5 are even more particularlypreferred. It is also understood in the art that the substitution oflike amino acids can be made effectively on the basis of hydrophilicity.As disclosed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine 5 (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitutions in a given OFA epitope are not limited to naturally andnon-naturally occurring amino acids. Certain mimetics that mimicelements of protein secondary structure may be used. The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins, including polyamino acids, exists chiefly toorientate amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is thus designed to permit molecular interactions similar to thenatural molecule. Some successful applications of the peptide mimeticconcept have focused on mimetics of β-turns within proteins, which areknown to be highly antigenic. Likely α-turn structure within apolypeptide can be predicted by computer-based algorithms. Once thecomponent amino acids of the turn are determined, mimetics can beconstructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains.

In addition to the 20 “standard” amino acids provided through thegenetic code, modified or unusual amino acids as shown in table 1 canalso be used in the present invention.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid Et-Asn N-Ethylasparagine bAad 3-Aminoadipicacid Hyl Hydroxylysine bAla beta-alanine, beta- aHyl allo-HydroxylysineAminopropionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu4-Aminobutyric Acid, 4Hyp 4-Flydroxyproline piperidinic acid Aep6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid alleallo-Isoleucine Aib bAib 2-Aminoisobutyric acid MeGly N-Methylglycinesarcosine 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpr2,3-Diaminopropionic acid Om Omithine EtGly N-Ethylglycine

The OFA epitopes of the present invention may be administered to treator prevent any cancer in a mammal that is characterized by the presenceof OFA. Tc cells recognize OFA epitopes bound to class I MHC molecules.In view of the variability in the MHC proteins from patient to patient,and the multitude of Tc clones, it is preferred to formulate a complexor “cocktail” of OFA epitopes that stimulate different clones of Tccells. In some embodiments of the present invention, the compositioncontaining the Tc epitope(s) may also contain one or more Th epitopes.Th cells recognize OFA epitopes that are bound to MHC class II proteins,which again may vary from patient to patient. The stimulation of bothCD4+ Th cells and CD8+ Tc cells may provide a greater immunogenic effectthan the use of Tc epitope(s) alone. See, Zeng, J. Immunother.24:195-204 (2001). The cocktail may be administered in several forms. Insome embodiments, they are formulated as a mixture of peptides. In otherembodiments, two or more epitopes are linked together to form a longerpolypeptide that is amenable to synthetic (non-recombinant) synthesise.g., a 20-60mer, thus including from about 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, to about 60amino acids. In some preferred embodiments, the polypeptide containsabout 50 amino acid residues. In these embodiments, it is preferred touse a linking agent that may be peptidic (e.g., about 3-5 amino acids inlength) or non-peptidic in nature (e.g., a disulfide bridge).Determination of effective and optimal spacing between individualepitopes for purposes of binding with MHC proteins and identification byantigen processing cells may be determined in accordance with standardtechniques.

In embodiments involving multiple OFA epitopes, administration can befacilitated by linking them to a common core structure such as amulti-branched lysine or arginine core to induce peptide specific CTLresponses. (Tam, PNAS USA 85:5409-5413 (1988); Posnett et al., J. Biol.Chem. 263:1719-1725 (1988)). Thus, in these embodiments where aplurality of Tc (and optionally one or more Th epitopes) areadministered, e.g., contained in a given complex or cocktail, thecomposition will contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,etc.) of the Tc epitopes e.g., epitopes containing OFA (53-60), OFA(81-88), OFA (148-155), OFA (156-163) and OFA (229-236), and optionallyone or more, and preferably two or more Th epitopes e.g., epitopescontaining OFA (152-161), OFA (229-238), OFA (241-250), OFA (253262),OFA (277-286) and OFA (285-294).

In yet other embodiments, the cocktail of epitopes is in the form of anOFA derivative that contains epitopes that stimulate Tc cells (andoptionally one or more epitopes that stimulate Th cells) but that lackepitopes that stimulate Ts cells. For instance, an OFA derivative maydiffer from intact OFA in that it lacks any one or more, and preferablyall Ts epitopes containing OFA (17-24), OFA (37-44), OFA (97-104) andOFA (140-147). Thus, the derivative may lack an entire region thatcontains multiple (overlapping and distinct) epitopes that stimulate Tscells or it may lack one or more individual epitopes (e.g., those whichhave the highest affinity for Ts cells out of the epitopes that residewithin the region of OFA). Regardless of the manner in which the OFAepitopes are administered, internalization and cellular processing ofthe epitopes in dendritic cells and presentation of antigenic peptide onthe cell surface would be expected as shown in other systems. See, Ota,et al., Cancer Res. 62:1471-1476 (2002); Mattner, et al., Cancer Res.62:1477-1480 (2002). Thus, the OFA epitopes may be used to inhibitgrowth or proliferation of a type of cancer, through enhancing aprotective immune system response to OFA-bearing tumor cells (caused bypreferential stimulation of Tc optionally with Th1 cells, relative to Tscells), or to induce cancer immunity. Cancer therapy in accordance withthe present invention may be evaluated by monitoring the production ofcytokines by the peripheral blood lymphocytes of the patient. One suchmethod entails cytokine enzyme-linked immunospot (ELISPOT) assays inconjunction with computer-assisted image analysis after short in-vitrostimulation of the spleen cells (lymphocytes). Cytokines of interestinclude interferon-gamma, tumor necrosis factor alpha, IL-4, IL-5 andIL-10. For example, abnormally high levels of IL-10 would likely beindicative of relatively (and undesirably) high levels of CD8 Tsuppressor cells, indicative of immune suppression. On the other hand,immune enhancement and effective therapy would likely be evidenced byhigh levels of CD8 cells secreting interferon gamma and tumor necrosisfactor alpha.

The OFA epitopes and derivatives of the present invention may beproduced using a solid-phase peptide synthesis technique or viarecombinant DNA technology by incorporating a DNA encoding the epitopeinto an appropriate expression vector, transforming a host via thevector, culturing the host (typically a bacterium such as E. coli) andisolating the expression product therefrom. The DNA sequence of fulllength OFA is attached hereto. Thus, it contains the sequences encodingthe epitopes. Persons skilled in the art will appreciate thatpolynucleotides encoding the epitopes other than the sequences disclosedherein can be prepared e.g., to accommodate codon preference of a givenhost, in view of the degeneracy of the genetic code. See, e.g., Watson,et al., Recombinant DNA, 2nd Ed., Freeman, N.Y. (1993). Likewise, thenucleotide sequence of a given OFA derivative may be easily prepared(e.g., by deleting from the full length sequence the sequences encodingthe aforementioned regions or epitopes). Synthetic schemes arepreferred. The OFA analogs may be purified in accordance with standardtechniques such as reverse-phase HPLC.

The OFA epitopes of the present invention may be formulated intopharmaceutical preparations for administration via any desired routee.g., subcutaneously, intravenously or intramuscularly, althoughintradermally or mucosally is preferred. Intradermal and mucosaladministration are advantageous from the standpoints of lower doses andrapid absorption respectively. Mucosal routes of administration includeoral, rectal and nasal administration. Preparations for mucosaladministrations are suitable in various formulations as described below.The route of administration can be varied during a course of treatment.Variables such as dosage amounts, and timing and mode of administrationwill vary depending on several factors including the weight and overallhealth of the patient as well as the state of the disease. In someembodiments, the OFA analogs are administered in an amount of from about5 μg to about 50 μg, given every two weeks for about 6½ weeks.

If the OFA epitope is water-soluble, it may be formulated in anappropriate buffer such as phosphate buffered saline or otherphysiologically compatible solutions. If on the other hand, the OFAanalog has poor solubility in aqueous solvents, it may be formulatedwith a non-ionic surfactant such as TWEEN®, or polyethylene glycol.Thus, the OFA epitopes may be formulated for administration byinhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral, rectal administration or, in the case oftumors, directly injected into a solid tumor. For oral administration,the pharmaceutical preparation may be in liquid form, for example,solutions, syrups or suspensions, or may be presented as a drug productfor reconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The pharmaceutical compositions may take the form of, forexample, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Preparations fororal administration may be suitably formulated to give controlledrelease of the OFA analog(s). Such compositions-may take the form oftablets or lozenges formulated in conventional manner.

For administration by inhalation, the OFA epitopes may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the OFA epitopes and a suitable powder basesuch as lactose or starch.

The OFA epitopes may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. The complexes mayalso be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides. In addition to the formulationsdescribed previously, the OFA epitopes may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the OFA epitopes may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Micelles, liposomes and emulsions are well known examples ofdelivery vehicles or carriers for hydrophilic drugs, and are suitabledelivery vehicles for the OFA epitopes of the present invention. Thecompositions may, if desired, be presented in a pack or dispenser devicethat may contain one or more unit dosage forms containing thenoncovalent complexes. The pack may for example comprise metal orplastic foil, such as a blister pack. The pack or dispenser device maybe accompanied by instructions for administration.

The immunogenic or immunotherapeutic compositions of the presentinvention contain a carrier in which the OFA/iLRP peptides can besuspended and, in general, allow a slow release of the OFA/iLRP toinduce a longer period of immunization. The immunogenic compositions ofthe present invention will also typically contain an adjuvant. Inpreferred embodiments, the carrier also functions as an adjuvant.Freunds adjuvant (IFA) has been used in human immunotherapy againstmelanoma involving gp 100 peptide immunization (Rosenberg, S. A., J. C.Yang, D. J. Schwartzentruber, et al. 1998, Nat. Med. 4:321)). However,this adjuvant is not widely used in human vaccination protocols due toits undesirable side effects, such as erythema and induration at theinjection site. Microfluidized (MF) 59 is an emulsion consisting of 5%(v/v), squalene, 0.5% (v/v), Tween 80, and 0.5% (v/v) Span 85 in water.It has been reported that the addition of MF59 adjuvant emulsion toconventional subunit influenza antigen causes enhanced immunogenicitywithout any clinically significant increase of reactogenicity (R.Gasparini. T. Pozzi, E. Montomoli 2001, 17, 135-40). See, also Podda, A.2001, Vaccine 19:2673.

Unmethylated CpG dinucleotides in a certain base context (CpG motifs)contained in synthetic oligodeoxynucleotides (ODN) stimulate B cells andNK cells (Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, eta1.1995. CpG motifs in bacterial DNA trigger direct B-cell activation.Nature 374:546)). They also activate dendritic cells (DCs) and inducematuration of DCs into professional antigen presenting cells (APCs)(Sparwasser, T., E. S. Koch, R. M. Vabulas, et al. 1998. Eur. J.Immunol. 28:2045; Hartmann, G., G. J. Weiner, A. M. Krieg. 1999, Proc.Natl. Acad. Sci. USA 96:9305; Sparwasser, T., R. M. Vabulas, B. Villmow,et al. 2000, Eur. J. Immunol. 30:3591; Vabulas, R. M., H. Pircher, G. B.Lipford, et al. 2000, Immunol. 164: 2372), thereby enhancing theirability to stimulate antigen-reactive T cells in vitro and in vivo.ODN-containing CpG motifs (referred to as CpG ODN) also stimulatemacrophages to secrete Th1 cytokines, which are important in thedevelopment of a CTL response (Carson, D. A., E. Raz. 1997, 186:1621).In addition, CpG ODN have been shown to behave as adjuvant of Ab and CTLresponse directed against liposome-entrapped whole protein or classI-restricted peptides (Lipford, G. B., M. Bauer, C. Blank, et al. 1997.Eur. J. Immunol. 27:2340). Repeated administration of CpG ODNpotentiates the CTL response against CTL peptide or protein emulsifiedin IFA and promotes the survival in response to tumor challenge in bothprophylactic and therapeutic vaccination protocols (Davila, E., E.Celis. 2000, J. Immunol. 165:539). Evidence for the induction of aspecific CTL response against a CD8⁺ T cell peptide in the presence ofCpG ODN without additional adjuvant has been reported by assessing thecytolytic activity of lymph node cells after in vitro stimulation(Vabulas, R. M., H. Pircher, G. B. Lipford, et. al. 2000, J. Immunol.164:2372).

Cytokines such as fetal liver tyrosine kinase 3-ligand (Flt3-ligand orFL) that mobilize DCs in vivo will also expand various DC subsets invivo (Pulendran, B., J. L. Smith, G. Caspary, et al. 1999, Proc. Natl.Acad. Sci. USA 96:1036; Shurin, M. R., P. P. Pandharipande, T. D.Zorina, et al. 1997. Cell. Immunol. 179:174). FL has been shown toexpand distinct DC subsets in mice and to greatly augmentantigen-specific T and B cell responses against soluble antigens andtumors (Pulendran, B., J. L. Smith, M. Jenkins, et al. 1998, J. Exp.Med. 188:2075; Lynch, D. H., E. Andreasen, E. Maraskovsky, et al. 1997,Nat. Med. 3:625). Dendritic cells have a unique ability to stimulatenaive T cells. Recent evidence suggests that distinct DC subsets directdifferent classes of immune responses in vitro and in vivo. In humans,the monocyte-derived CD11c⁺ DCs induce T cells to produce Th1 cytokinesin vitro, whereas the CD11c⁻ plasmacytoid T cell-derived DCs elicit theproduction of Th2 cytokines. Administration of either flt3-ligand (FL)or granulocyte-colony stimulating factor (G-CSF) to healthy humanvolunteers dramatically increases distinct DC subsets, or DC precursors,in the blood. FL increases both the CD11c⁺ DC subset (48-fold) and theCD11c⁻ IL-3R⁺ DC precursors (13-fold). In contrast, G-CSF only increasesthe CD11c⁻ precursors (e.g., greater than 7-fold). Freshly sorted CD11c⁺but not CD11c⁻ cells stimulate CD4⁺ T cells in an allogeneic MLR,whereas only the CD11c⁻ cells can be induced to secrete high levels ofIFN-alpha in response to influenza virus. CD11c⁺ and CD11c⁻ cells canmature in vitro with GM-CSF+TNF-alpha or with IL-3+CD40 ligand,respectively. These two subsets up-regulate MHC class II co-stimulatorymolecules as well as the DC maturation marker DC-lysosome-associatedmembrane protein. In addition, they stimulate naive, allogeneic CD4⁺ Tcells. These two DC subsets elicit distinct cytokine profiles in CD4⁺ Tcells, with the CD11c⁻ subset inducing higher levels of the Th2 cytokineIL-10. The differential mobilization of distinct DC subsets or DCprecursors by in vivo administration of cytokines such as FL and G-CSFalso serves to manipulate immune responses in humans (B. Pulendran, etal., J. Immunol. 165:566-572 (2000)).

It has been further demonstrated that co-administration of type Iinterferon (IFN) with a human vaccine (influenza), causes a powerfuladjuvant effect, inducing a Th1-type of immune response and protectionagainst virus challenge (E. Proietti, et al., J. Immunol. 169:375-383(2002)). When given intramuscularly, type I IFN was far superior to alumand was equivalent to complete freund's adjuvant (CFA), considered oneof the most powerful adjuvants in animal models, as well as to MF59.

Yet other adjuvants contain polyinosinic acid-polycytidylic acid(poly(I-C)). The effect of this adjuvant on DC expression of IL-15 aswell as the capacity of IL-15 to serve as a DC activator has beenreported in Mattei, et al., J. Immunol. 167:1179-1187 (2000). Injectionof poly(I:C) into mice induces up-regulated expression of both IL-15 andIL-15R alpha by splenic DCs. In addition, IL-15 treatment enhanced theexpression of costimulatory markers on DCs, as well as their ability tostimulate antigen-specific CD8⁺ T cell proliferation. Further, IFN-gammasecretion by splenic DCs was markedly increased after treatment withIL-15, suggesting that IL-15 modulates the ability of DCs to polarize Tcell responses.

It is also preferred that the carrier contains an agent that activates(and thus causes maturation of) dendritic cells for optimal presentationof the OFA/iLRP peptides to T cells. The adjuvant may possess thisproperty. As described above, unmethylated CpG oligodeoxynucleotides andpoly (I:C) serve that purpose. Bacterial peptidoglycan andlipopolysaccharide activate dendritic cells as well. However, they needto be isolated and purified from bacteria. Thus, the methylated CpGoligodeoxynucleotides or polyinosinic acid:polycytidylic acid, arepreferred for this purpose because as chemically synthetic carriers,they will activate dendritic cells so they can optimally present theOFA/iLRP peptides present in the carrier to T cells without having apotential disadvantage from the standpoint of microbial contamination.

As disclosed above, liposomes are suitable delivery vehicles for the OFAepitopes and derivatives of the present invention. Liposomes composed ofnatural or synthetic ester phospholipids (conventional liposomes) areknown to be effective as immuno-adjuvants and as vaccine carriers(White, et al., Vaccine 13:1111-1122 (1995); Guan, et al., BioconjugateChem. 9:451-458 (1998)). A liposome-based vaccine against hepatitis Ahas been licensed for human use (Ambrosch, et al., Vaccine 15:1209-1213(1997)). Sterically stabilized cationic liposomes (SSCL) have been usedto significantly enhance the therapeutic efficacy of CpG ODN byincreasing the bioavailability and duration of action of CpG ODN.Encapsulating CpG ODN in sterically stabilized cationic liposomesprovides protection from serum nucleases while facilitating uptake by Bcells, dendritic cells and macrophages. In an immunization model,coencapsulation of CpG ODN with protein antigen (Ag) magnified theAg-specific IFN-gamma and IgG responses by 15- to 40-fold compared withAg plus CpG ODN alone (Gursel, et al., J. Immunol. 167:3324-3328(2001)).

There have been a number of approaches to improve the immuno-adjuvantaction of liposomes, some of which involve modification of the liposomestructure. Small size and positively charged carriers have been shown tobe preferentially taken up by phagocytic cells such as DCs/macrophagesand to elicit a significant CTL response. The mechanisms by which theliposomally encapsulated protein/peptide antigens are directed to thecytosol are believed to result from passive escape of the antigen fromthe endosomes into the cytoplasm where they access the MHC class Iprocessing pathway (Zhou et al., Immunobiology 190:35-52 (1994)).However, a peptide sequence, referred to as antennapedia homeodomain(AntpHD), can effectively introduce CTL epitopes into the class Iprocessing pathway and induce CTL in vivo. Chikh, et al., J. Immunol.167:6462-6470 (2001), describes a vaccine which uses a recombinantpeptide consisting of a CTL epitope, which binds MHC class I molecules,and a peptidic vector, AntpHD, that can deliver peptides into thecytosol of cells, where it is processed by the proteasome complex. Theincrease of the CTL response induced by AntpHD-fused peptide inliposomes correlates with this active transport to class I-processingpathway. Moreover, addition of CpG ODN immunostimulatory sequencesfurther increase the CD8⁺ T cell response. This strategy combininglipid-based carriers with antpHD peptide to target poorly immunogenicAgs into the MHC class I processing pathway represents a plausibleapproach for CTL vaccines that may have important applications fordevelopment of cancer vaccines. Further, the unique ether glycerolipidsof Archaea can be formulated into vesicles (archaeosomes) with strongadjuvant activity for MHC class I and class II presentation (Krishnan,J. Immunol. 165:5177-5185 (2000)). These investigators found thatimmunization of mice with ovalbumin (OVA) entrapped in archaeosomesresulted in a potent Ag-specific CD8⁺ T cell response, as measured byIFN-gamma production and cytolytic activity toward the immunodominantCTL epitope OVA (aa 257-264). Interestingly, a long-term CTL responsewas generated with a low Ag dose even in CD4⁺ T cell deficient mice,indicating that the archaeosomes could mediate a potent T helpercell-independent CD8⁺ T cell response. Thus, delivery of proteins inself-adjuvanting archaeosomes represents a useful strategy for targetingexogenous antigens to the MHC class I pathway for induction of CTLresponse. Thus, several types of vesicles are useful as carriers for theimmunotherapeutic agents of the present invention.

The OFA epitopes may be administered without an adjuvant. In certainembodiments, the epitopes are attached or conjugated to a lipophilicgroup and administered as a lipopeptide vaccine. See, Gahery-Segard, etal., J. Virol. 74:1694-1703 (2000); Gras-Masse, Mol. Immunol. 38:423-431(2001); Vitello, et al., J. Clin. Invest. 95:341-349 (1995); BenMohamed,et al., Immunology 106:113-121 (2002); and Schild, et al., J. Exp. Med.174:1665-1668 (1991) (reporting that an influenza virus lipopeptidewithout additional adjuvant elicited influenza virus-specific cytotoxicT (Tc) responses whereas the corresponding peptide without a lipidmoiety did not). Examples of lipophilic groups includeN-epsilon-palmitoyl-L-lysylamide and α-aminohexandecanoic acid. Peptidescovalently attached to the N-epsilon-palmitoyl lysine moiety have beenshown to activate macrophages and induce secretion of pro-inflammatorycytokines IL-1, IL-6, and TNF-α (Rouaix, et al., Vaccine 12:1209-14(1994)). Lipopeptides also appear to target dendritic cells (Tsunoda, etal., Vaccine 17:675-685 (1999), which reported that in a comparativestudy with a bipalmitoylated peptide and its non-lipidic peptideanalogue, immunohistological analysis of tissue from immunized micerevealed both macrophages and dendritic cell-associated lipopeptide, butnot its non-lipidic analogue, and implicated dendritic cells inprocessing and presentation of lipopeptide particles to T cells).Dendritic cells, by contrast with macrophages, are unique in theircapacity to prime naive T cells against soluble antigens administered inthe absence of an adjuvant (Banchereau, et al., Nature 392:245-52(1998)). It has become increasingly clear that manipulation of theimmune response for vaccination purposes requires immunization routesallowing efficient antigen uptake by dendritic cells. (Mowat, Immunol.Lett. 65:133-40 (1999)). One study has shown that bone marrow-deriveddendritic cells take up a model lipopeptide more efficiently than domacrophages (BenMohamed, et al., The Lancet Infect. Dis. 2:425-31(2002)). Speculation is that this may be due to the palmitoyl moiety oflipopeptides fusing to lipid components of cell membranes andsubsequently delivering the lipopeptides into the cytoplasm of dendriticcells (BenMohamed, et al., Vaccine 18:2843-55 (2000); Andrieu, et al.,Eur. J. Immunol. 30:3256-65 (2000)). Besides binding and enteringdendritic cells for presentation, lipopeptides have been shown tointeract with Toll-like receptor 2 (Nishiguchi, et al., J. Immunol.166:2610-16 (2001)) on the dendritic cell and so induce dendritic cellmaturation which is required for optimal antigen presentation to Tlymphocytes.

Modification of a peptide by attachment to lipophilic molecules, such asN-epsilon-palmitoyl-L-lysylamide or α-aminohexadecanoic acid(mono-palmitoyl peptide) can be achieved by conventional methods ofpeptide synthesis and characterization. See, Loing, et al., J. Immunol.164:900-907 (2000); and Deprez, et al., Vaccine 14: 375-382 (1996). Forexample, the lipid tail may be attached a posteriori by chemoselectiveligation, which entails coupling of fully deprotected molecularfragments through two mutually and uniquely reactive functional groups.See, Gras-Masse, Mol. Immunol. 38:423-431 (2001). These approachesprovide for scalable manufacturing and low cost synthetic vaccines. Thelipopeptides produced by this methodology have been reported to induceas strong a CD8+ Tc cell response as the previously producedtri-palmitoyl lipopeptides (BenMohamed (2002), supra.). Also, suchlipopeptides, have been reported to induce CD4+ Th cell responses(Pialoux, et al., AIDS15:1239-49 (2001)). Mono-palmitoyl lipopeptideshave been reported to be tolerated by the host with no local reaction tothe synthetic lipopeptide vaccine (BenMohamed (2000), and Schild, etal., supra., and BenMohamed, et al., Immunol. 106:113-121 (2002)) inanimal models and in human volunteers (Seth, et al., AIDS Res. Hum.Retroviruses 16:337-43 (2000c)). This mono-palmitoyl approach,therefore, appears to offer unique advantages in safety, cost, purityand simplicity of construction and obviates the need for toxic vaccineadjuvants (Gupta, et al., Vaccine 13:1263-76 (1995)).

In preferred embodiments, the vaccine composition of the presentinvention contains a plurality (i.e., two or more) lipopeptides, each ofwhich contains a distinct Tc-inducing OFA epitope. In other preferredembodiments, the vaccine also contains one or more lipopeptides thatcontain a Th-inducing OFA epitope. The sequence of the epitopes willhave to be confirmed based on the HLA MHC proteins the patientexpresses. Administration, e.g., intradermal or subcutaneous injectionof this mixture of mono-palmitoyl-conjugated OFA/iLRP peptides will leadto uptake by and maturation of dendritic cells which then can presentthose peptides to Tc and Th cells in lymph nodes draining the site(s) ofimmunization. Thus, dendritic cells will be targeted in vivo by thelipopeptides.

There are many reasons why immunotherapy as provided by the OFA epitopesof the present invention is desired for use in cancer patients. First,if cancer patients are immunosuppressed, surgery with anesthesia andsubsequent chemotherapy may worsen the immunosuppression. Appropriateimmunotherapy in the pre-operative period using the compositions andmethods of the present invention may prevent or reverse theimmunosuppression. This could lead to fewer infectious complications andan accelerated wound healing. Second, tumor bulk is minimal followingsurgery; thus, immunotherapy is most likely to be effective in thissituation. Third, tumor cells tend to be shed into the circulation as aresult of surgery; thus, effective immunotherapy applied at this timecan eliminate these cells. Preventive and therapeutic utilities of thepresent invention are directed to enhancing the immunocompetence ofcancer patients before, during and/or after surgery, and to inducingtumor-specific immunity to cancer cells. While the ultimate clinicalobjective is total cancer regression and eradication, embodiments of thepresent invention are effective in inhibiting tumor growth andprogression of the disease. Compositions containing the OFA epitopes areuseful in the prophylaxis or treatment of cancer in mammals. The cancersinclude but not limited to human lymphomas, sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia and heavychain disease.

The therapeutic utility of the OFA epitopes and derivative of thepresent invention is not limited to in vivo uses. Mature dendritic cellsexpress high levels of class I and class II MHC molecules, as well ashigh levels of various costimulatory molecules. Thus, dendritic cellsare potent antigen-presenting cells for induction of Tlymphocyte-mediated immunity. They can efficiently present MHCprotein-bound antigenic peptides to T cells. The costimulatory moleculescan complete the activation signalling of the antigenic peptide:MHCprotein-induced T cells (Janeway, C. A., P. Travers, M. Walport, and M.Shlomchik. 2001. Immunobiology: The Immune System in Health and Disease.Garland Publishing, New York, pp. 307-309). In addition, the dendriticcells bring processed antigen from where it is encountered to the lymphnodes and spleen where immune responses are induced (Sallusto, et al.,Arthritis Res. 4 Suppl. 3:S127-132 (2002)). Pure dendritic cells can begenerated in vitro from a mammalian cancer patient's (e.g., human)peripheral blood monocytes (Nair, et al., Annals of Surgery 235:540-549(2002)). For example, culturing peripheral blood mononuclear cells(monocytes) from a patient for 7-8 days with GM-CSF and IL-4, will causedifferentiation of the monocytes into pure immature dendritic cells, andsubsequent culture in the presence of medium containing double-strandedpoly I:C RNA will induce dendritic cell maturation (Nair, supra., Holtl,et al., Clin. Cancer Res. 8:3369-76 (2002) (reporting on programming ofdendritic cells using tumor cell lysate)). If desired, the autologousmature dendritic cells can be cryogenically preserved in liquid nitrogenfor subsequent use with the patient from which they were derived.

The mature dendritic cells are cultured with one or more OFA/iLRPepitope(s) that specifically stimulate Tc cells, and optionally with oneor more epitopes that specifically stimulate Th cells under conditionssuitable to program the dendritic cells to potentiate T cell-mediated(anti-cancer) immunity e.g., for about 1 hour at about 37° C. The OFAepitopes can be added to the dendritic cells in medium or conjugated toa lipid adjuvant carrier (Zhou, et al., J. Immunother 25:289-303(2002)). The medium may also contain co-stimulants such as TNF-alpha,interleukins and prostaglandin. In general, the OFA epitope(s) are addedto the medium in microgram amounts.

After dendritic cell loading of the OFA/iLRP peptides, the patient isadministered the loaded dendritic cells. In one embodiment, the patientis administered about 3×10⁷ peptide-loaded dendritic cells via i.v.injection over 2-3 minutes, followed by intradermal administration ofabout 1×10⁶ OFA/iLRP epitope-loaded autologous dendritic cells in avolume of about 0.1 ml autologous plasma into the volar aspect of theforearm or thigh, every 4 weeks for four immunizations. Persons skilledin the art will appreciate that many variations of this treatmentregimen would also be useful. A single dose of the loaded dendriticcells may provide a therapeutic benefit. Even if epitopes that arerecognized by Ts cells are inadvertently present, dendritic cellpresentation in vivo may actually overcome induction of thosesuppressive cells. Dendritic cell presentation of an autologous nuclearantigen actually breaks self-tolerance with activation of Th1 immunityand IgG antibody production in mice (Suen, et al., Immunol. 106:326-335(2002)). Thus, immunization with OFA/iLRP epitope-loaded autologousmature dendritic cells may potentiate the immunity achieved with theimmunotherapeutic compositions of the present invention alone. Theinvention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to limit the scope of theinvention described herein.

EXAMPLES Summary of iLRP Epitopes Recognized by CD8⁺ Tc and Ts and CD4⁺Th1 Clones from BALB/c Mice, and Experimental Protocol

The epitopes recognized by 15 iLRP-reactive T cell clones derived fromeither naive BALB/c mice, BALB/c mice bearing MCA1315 fibrosarcomatumors, or from BALB/c mice immunized with either 1 or 10 μg ofrecombinant murine iLRP were determined by testing the proliferation ofthose clones to overlapping peptides spanning the region of the iLRPmolecule recognized by each clone in the presence of IL-2, as a growthfactor for T cells and irradiated, T cell-depleted, syngeneic spleencells as antigen-presenting cells. Proliferation was measured throughELISA determination of 5-bromodeoxyuridine incorporation during culture.

More specifically, the epitope-specificity of the iLRP-specific Th1, Tc,and Ts clones was determined using a modification of the5′-bromodeoxyuridine (BudR) incorporation ELISA technique described inRohrer, J. W., A. L. Barsoum, D. L. Dyess, J. A. Tucker, and J. H.Coggin, Jr. 1999. Human breast carcinoma patients develop clonableoncofetal antigen-specific effector and regulatory T lymphocytes. J.Immunol. 162:6880, using the Biotrak BUdR incorporation assay (Amersham,Arlington Heights, Ill.). Briefly, at the time of restimulation of theclones, a portion of the cloned T cells were assayed for proliferationto antigen-presenting cells (APC) and various iLRP peptides. The assaywas performed with 10,000 viable cloned T cells/well plus 105 irradiatedsyngeneic spleen cells (APC) plus 100 ng/well of intact iLRP/OFA proteinor the various truncated iLRP/OFA proteins or the various iLRP/OFApeptides in Iscove's modified Dulbecco's medium (IMDM) containing 2 mML-glutamine, 100 U/ml of penicillin G, 100 μg of streptomycin sulfate,and 10% fetal calf serum. The cells were cultured for a total of 48hours. After 24 hours of culture 5′-bromodeoxyuridine was added to afinal concentration of 10 μM/well. The cells were cultured for another24 hours. At the end of the last 24 hours of incubation, the plates werecentrifuged at 300×g for 10 min, and the labelling medium removed. Thecells were then dried at 60° C. for 1 hour. The cells were fixed with anethanol fixative provided in the Biotrak kit for 30 min at roomtemperature, fixative was removed, and the wells were coated withblocking buffer (1% protein in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) andincubated for 30 min at room temperature. The blocking buffer wasremoved, 100 μl of 1:100 diluted peroxidase-labelled anti-BUdR antibodywas added to each well and the plates were incubated for 90 min at roomtemperature. The antibody solution was removed, and the wells washedthree times with 300 μl/well of wash buffer. 200 μl of3,3′,5,5′-tetramethylbenzidine in 15% (v/v) DMSO was added to each welland the plate was covered and incubated at room temperature whileoscillating gently for 5-30 min. When the required color density wasreached, the reaction was stopped by adding 25 μl of 1M sulfuric acid toeach well and the plate read on a microELISA reader at 450 nm.

Each time the assay was done, clones were stimulated by APC and intactiLRP/OFA as a positive control. Cloned T cells were also cultured in thepresence of only APC in IMDM or in the presence of APC and an iLRP/OFApeptide or truncated iLRP/OFA known not to stimulate the clones beingtested. These served as negative controls. In wells where BUdR wasincorporated (the iLRP/OFA stimulates proliferation of the T cells), theA450 was at least 10-fold and often approximately 50-fold higher thanthe negative controls. Because the Ts cells always presented withapparently low affinity T cell antigen receptors, their BUdRincorporation gave A450 values about 10 times higher than the negativecontrols (e.g., about 0.2 vs. 0.02), but about 5 times lower than theTh1 or Tc clones (e.g., usually about 0.9).

Determination of the portion of the truncated iLRP/OFA protein orpeptide reacted to by a given clone was done by analysis of theproliferation pattern to the various truncated proteins or peptides. Itwas then determined what amino acid sequence was shared by thoseiLRP-truncated proteins or peptides that stimulated a T cell clone toproliferate.

Table 2 below shows the distribution of epitopes for the various typesof T cell clones established, deduced on the basis of maximal response.Even though the cytotoxic T (Tc) cells and the IL-10-secreting Ts cellsare both CD8 T cells and are both class I MHC-restricted, these twotypes of T cells recognize distinct epitopes on OFA. (SEQ ID NOS 6, 7,9, 10, 8, 22-25, 25, and 15-20, respectively, in order of appearance)

TABLE 2 iLRP Epitope iLRP-specific Amino Acid iLRP Epitope T Cell CloneT Cell Type Region Sequence M3 Tc 53-60 RTWEKLLL L6 Tc 81-88 NTGQRAVL L5Tc 148-155 CNTDSPLR L4 Tc 156-163 YVDIAIPC L2 Tc 229-236 GEWTAPAP L1 Ts17-24 KLLAAGTH H5 Ts 37-44 YIYKRKSD M11 Ts  97-104 TPIAGRFT H2 Ts140-147 VNLPTIAL H4 Ts 140-147 VNLPTIAL H3 Th1 152-161 SPLRYVDIAI NC1Th1 229-238 GEWTAPAPEF L3 Th1 241-250 AQPEVADWSE M2 Th1 253-262QVPSVPIQQF H1 Th1 277-286 SAAPTAQATE NC4 Th1 285-294 TEWVGATTDW

Specificity Analysis of Various Types of iLRP-Reactive T Cell Clones

Specificity of T_(s) clone L1 using N-terminal peptides Overlapping12mer peptides of the first 25 amino acids of murine iLRP were produced.Thus, peptides 1-4 corresponded to amino acid residues 1-12, 5-16, 9-20and 17-28 of murine iLRP. L1 is a BALB/c mouse CD8 Ts clone that wasestablished from spleens of mice immunized twice at 2-week intervalswith OFA/iLRP-conjugated nitrocellulose particles (i.e., a totalOFA/iLRP dose each i.p. injection of 1 mcg). The spleens were harvested2 weeks after the last immunization and the spleen cells were minced andwashed by centrifugation, then cultured with 10⁵ irradiated MCA1315fibrosarcoma tumor cells for two weeks in the presence of IL-2, IL-6,and interferon-gamma. The cells were then cloned by limiting dilution at0.2 tumor-reactive T cells/well in the presence of 10⁵ irradiated,syngeneic spleen cells and 10⁵ irradiated, syngeneic MCA1315fibrosarcoma cells in medium containing recombinant murine IL-2,recombinant murine IL-6, and recombinant murine IFN-gamma using themethod described in Rohrer et al., 1995, J. Immunol. 154:2266. Resultsare shown in Table 3. (SEQ ID NOS 26-29, respectively, in order ofappearance)

TABLE 3 BUdR Incorporation (A₄₅₀) of iLRP-specific T cell clone L1 cellsafter exposure to various iLRP-derived peptides. iLRP a. a. Stimulantsequence Expt. 1 Expt. 2 Medium .007 .009 iLRP truncated a. a.'s 242-295.016 .014 protein 13 intact iLRP a.a.'s 1-295 .20 .23 Peptide 1NSGALDVLQMKE .018 .020 Peptide 2 LDVLQMKEEDVL .017 .019 Peptide 3QMKEEDVLKLLA .09 .07 Peptide 4 KLLAAGTHLGGT .28 .27

Clone L1 proliferates to a peptide contained almost entirely in peptide4, but does react somewhat to peptide 3 as well, but a tetrapeptide thatis common to both. The epitope that will maximally stimulate the L1clone is deduced as KLLAAGTH (SEQ ID NO: 22). Results of an analysis ofthe specificity of iLRP-specific T cell clones reactive to iLRP peptidespanning amino acid residues 26-61 are shown in Table 4.

TABLE 4 CD8 T Cell Clones Stimulant M3 (Tc) H5 (Ts) Medium .008, .009.005, .007 P15 (aa 81-92) .018, .017 .017, .018 intact iLRP  .27, .30  .23, .26  P1 (aa 25-36) .017, .02  .017, .016 P2 (aa 29-40)  .02, .021.016, .018 P3 (aa 33-44) .017, .019  .23, .25  P4 (aa 37-48)  .02, .018 .22, .25  P5 (aa 41-52) .018, .021 .017, .018 P6 (aa 45-56) .017, .021.021, .015 P7 (aa 49-60)  .25, .28  .019, .016 P8 (aa 53-64)  .27, .30 .017, .018 P9 (aa 57-68) .018, .021 .017, .016

It appeared that clone M3 was responding to an epitope between aminoacids 49 and 64 while clone H5 was responding to an epitope betweenamino acids 33 and 48. Once again, it appeared that distinct epitopeswere being seen by Tc and Ts clones.

Analysis of Specificity of iLRP-specific T Cell Clones Reactive to iLRPPeptide Spanning Amino Acids 62-135

The same proliferation assay as described above was performed with CD8cytotoxic T cell clone L6 and Ts clone M11. The results of theproliferation assay for clone L6 to iLRP deletion mutant truncatedproteins showed that it recognized an epitope between amino acids 62 and135 while the proliferation assay results to iLRP deletion mutanttruncated proteins of clone M11 showed it responded to some epitopecontained between amino acids 81 and 135. The proliferation assayresults of these two clones to iLRP peptide 12-mers spanning the 62-135amino acid region was conducted to define the epitopes for each cloneand to determine if once again, the Tc and Ts cells recognized distinctepitopes. The results are shown in Table 5, and the deduced epitopes ofthe various clones are shown in Table 6.

TABLE 5 CD8 iLRP-specific T Cell Clones Stimulant L6 (Tc) M11 (Ts)Medium .007, .008 .006, .008 P3 (33-44) .016, .017 .015, .014 IntactiLRP  .95, .96   .21, .19  P10 (aa 61-72) .017, .017 — P11 (aa 65-76).015, .016 — P12 (aa 69-80)  .02, .015 — P13 (aa 73-84) .016, .018 — P14(aa 77-88)  .96, .94  .015, .017 P15 (aa 81-92)  .95, .92  .015, .016P16 (aa 85-96)  .02, .017 .014, .017 P17 (aa 89-100) .015, .017 .015,.016 P18 (aa 93-104) .017, .02   .21, .18  P19 (aa 97-108) .015, .014 .23, .15  P20 (aa 101-112) .016, .018 .017, .02  P21 (aa 105-116)  .02,.017 .017, .014 P22 (aa 109-120)  .02, .015 .014, .016 P23 (aa 113-124).015, .016 .015, .016 P24 (aa 117-128) .016, .017 .015, .017 P25 (aa121-132) .017, .016 .017, .015 P26 (aa 125-136) .016, .015 .015, .018(SEQ ID NOS 30-37, resdectively, in order of appearance)

TABLE 6 iLRP Peptides That Induced T Cell Clone Proliferation P3 (aa33-44) QMEQYIYKRKSD P4 (aa 37-48) YIYKRKSDGIYI P7 (aa 49-60)INLKRTWEKLLL P8 (aa 53-64) RTWEKLLLAARA P14 (aa 77-88) ISSRNTGQRAVL P15(aa 81-92) NTGQRAVLKFAA P18 (aa 93-104) ATGATPIAGRFT P19 (aa 97-108)TPIAGRFTPGTF

Clone H5 (a Ts clone) proliferated to iLRP peptides 3 and 4 equallywell. The common sequence in those peptides is YIYKRKSD (SEQ ID NO: 23)(amino acids 37-44). Therefore, it was deduced that clone H5 recognizesthat 8 amino acid iLRP epitope presented by a class I H-2d MHC protein.

Clone M3 (a Tc clone) proliferated to iLRP peptides 7 and 8 equallywell. The common sequence in those peptides is RTWEKLLL (SEQ ID NO:6)(amino acids 53-60). Therefore, it was deduced that that 8 amino acidsequence is the epitope presented by a class I H-2d MHC protein that isrecognized by Tc clone M3. M3 must have a low affinity TCR because itproliferated no better to iLRP than did the Ts clones.

Clone L6 (a Tc clone) proliferated equally well to iLRP peptides 14 and15. The common sequence of those peptides is NTGQRAVL (SEQ ID NO:7)(amino acids 81-88). Therefore, it was deduced that that 8 amino acidsequence is the iLRP epitope presented by a class I H-2d MHC proteinthat is recognized by Tc clone L6.

Clone M11 (a Ts clone) proliferated equally well to ILRP peptides 18 and19. The common sequence of those peptides is TPIAGRFT (SEQ ID NO: 24)(amino acids 97-104). Therefore, it was deduced that that 8 amino acidsequence is the iLRP epitope presented by a class I H-2d MHC proteinthat is recognized by Ts clone M11.

The question was posed whether clones which appear to be specific for aepitopes contained between amino acids 136 and 166 respond byproliferation to antigen-presenting cells presenting the processed 30merwhich has the sequence of a.a.'s 136-166 of iLRP. To answer thisquestion, the proliferation assay was done as above except that clonesH3 (Th1), H2 and H4 (T_(s)), and L4 and L5 (T_(c)) were used. Theresults are shown in Table 7.

TABLE 7 BUdR incorporation (A₄₅₀) of iLRP-specific clones deductivelydetermined to be reactive to epitopes within a.a.'s 136-166 whenstimulated by that sequence or controls. Truncated iLRP iLRP protein 13peptide (a.a.'s (a.a.'s Clone Medium 242-295) intact iLRP 136-166)  H3(Th1) .008 .017 .94 91 H2 (Ts) .008 .015 .26 .24 H4 (Ts) .008 .016 .23.21 L4 (Tc) .007 .017 .89 .90 L5 (Tc) .008 .015 .92 .87

The clones whose reactivity patterns to the various truncated iLRPproteins suggested a specificity within amino acids 136-166, proliferateto the 30mer peptide which is the sequence of a.a.'s 136-166(EASYVNLPTIALCNTDSPLRYVDIAIPCNNK) (SEQ ID NO: 38). Although there wasapproximately a 4.5-fold difference between the BudR incorporation ofthe Th1 or Tc clone and the Ts clones, the data for the Ts clone aregreater than 10-fold higher than the irrelevant iLRP peptide induced inany of the clones. The difference between the proliferation of the Th1or Tc and the Ts clones was probably due to the T cell antigen receptoraffinity for iLRP:self MHC. In previous experiments, using 75-100ng/well of iLRP/OFA protein to stimulate proliferation, one dose wasnear the plateau of the dose response for the high affinityreceptor-bearing Th1 and Tc clones, but just barely able to inducemeasurable proliferation (³H-thymidine- or BUdR-incorporation) of thelow-affinity receptor-bearing T cells (which was composed by some of theTh1 and Tc clones and all of the IL-10-secreting Ts clones).

Analysis of Additional Regions of Murine iLRP: Identification ofEpitopes Contained Between Amino Acids 136 and 166

The BUdr incorporation assay for proliferation described for the assaysabove was used. The clones H3, H2, H4, L4, and L5 which proliferate inresponse to x-irradiated, syngeneic spleen cell-presented 30-mer iLRPpeptide 136-166 in the presence of 100 U/ml of recombinant murine IL-2,were tested for their proliferation to overlapping 12-mer peptidescovering the amino acid sequence of iLRP peptide 136-166. The resultsand the peptides used in the experiments are shown in Tables 8 and 9respectively.

TABLE 8 BudR Incorporation (A₄₅₀) of T cell clones specific for iLRPpeptide 136-166 to various peptides spanning that portion of iLRPprotein. Stimulus Trun- cated iLRP iLRP iLRP iLRP iLRP iLRP iLRP ProteinIntact peptide peptide peptide peptide peptide peptide 13 iLRP 31-1 31-231-3 31-4 31-5 31-6 T Cell (242- Protein (136- (140- (144- (148- (152-(156- Clone IMDM 295) (1-295) 147) 151) 155) 159) 163) 167) H3 .007 .015.88 .016 .015 .016 .81 .95 .03 (Th1) H2 .008 .017 .20 .23 .22 .015 .016.016 .015 (Ts) H4 .008 .018 .21 .22 .24 .017 .016 .016 .016 (Ts) L4 .009.017 .89 .015 .018 .016 .023 .91 .90 (Tc) L5 .007 .016 .90 .017 .016 .90.89 .024 .015 (Tc)

TABLE 9 (SEQ ID NOS 39-44, respectively, in order of appearance) AminoAcid Sequence of Overlapping 12-mer Peptides of iLRP 136-166 PeptideiLRP 12-mers a.a. Range a.a. Sequence 31-1 136-147 EASYVNLPTIAL 31-2140-151 VNLPTIALCNTD 31-3 144-155 TIALCNTDSPLR 31-4 148-159 CNTDSPLRYVDI31-5 152-163 SPLRYVDIAIPC 31-6 156-167 YVDIAIPCNNKG

The Tc clone L4 proliferated to peptides 31-5 and 31-6 equally, so itwas deduced that the sequence YVDIAIPC (SEQ ID NO: 10), which is commonto both peptides, is an epitope specifically recognized by that clone oniLRP. Similarly, the equal proliferative response of Tc clone L5 topeptides 31-3 and 31-4 strongly suggested that an epitope specificallyrecognized by this clone is the sequence common to those two peptides(i.e., CNTDSPLR) (SEQ ID NO: 9). The Ts clones H2 and H4 bothproliferated equally to peptides 31-1 and 31-2. Thus, it was deducedthat both of those clones specifically recognize the 8-amino acidsequence common to those 2 peptides, namely VNLPTIAL(SEQ ID NO: 25).Regarding Th1 clone H3, the strongest response was to peptide 31-5 witha slightly lower response to 31-4. Therefore, it was deduced that theepitope having the sequence SPLRYVDIAI(SEQ ID NO: 15) was specificallyrecognized by clone H3. This sequence is entirely present in peptide31-5, but peptide 31-4 lacks the last two amino acids of that peptide.The deduced epitopes that provide maximal stimulation to the T cellclones tested are set forth in Table 10.

TABLE 10 (SEQ ID NOS 15, 25, 25, 10 & 9, respectively, in order ofappearance) Proposed iLRP Epitopes for iLRP Peptide 136-166-reactive TCell Clones T Cell Clone Proposed iLRP Epitope H3 (Th1) SPLRYVDIAI (a.a.152-161) H2 (Ts) VNLPTIAL (a.a. 140-147) H4 (Ts) VNLPTIAL (a.a. 140-147)L4 (Tc) YVDIAIPC (a.a. 156-163) L5 (Tc) CNTDSPLR (a.a. 148-155)

Initially, 1 Th1, 2 Ts, and 2 Tc clones appeared to all respond toepitopes contained in the 30-mer iLRP peptide composed of amino acids136-166. However, once the region was further analyzed using 12merpeptides spanning that 30mer region, distinct epitopes for theregulatory T cells and the effector T (Th1 and Tc) cells were found. Thetwo Ts clones both responded to an epitope composed of amino acids140-147 while the two Tc clones responded to epitopes composed of aminoacids 148-155 and 156-163, respectively. The Th1 clone responded to anepitope that bridged the two Tc epitopes (amino acids 152-161). Thus, itwas surprising to find the immuno-regulatory Ts clones responded todifferent epitopes than the Tc cells even though both types of cells areCD8, class I MHC-restricted iLRP-specific clones.

Analysis of Specificity of iLRP-Specific Th1 Cell Clone Reactive to iLRPPeptide Spanning Amino Acids 168-242

As before, proliferation assays using ELISA measurement of BudRincorporation to measure proliferation of T cells cultured with variousiLRP peptides and T cell-depleted, irradiated, syngeneic spleen cells asantigen presenting cells, all in medium containing 100 U/ml of IL-2 weredone. The clones were with antigen-presenting cells and 100 ng/well ofan irrelevant iLRP peptide as a negative control, intact iLRP protein asa positive control, or overlapping 12-mer iLRP peptides that spanned the168-242 region of OFA. After 24 hours, BudR was added and the culturecontinued for another 24 hours, at which time, the cells were harvestedand assayed for BudR incorporation as per the instructions of BioTrakCell Proliferation ELISA System. The data presented below in Table 11are the A₄₅₀ readings on the wells after the assay was complete. OFAepitopes are shown in Table 11a.

TABLE 11 Proliferation Results of Th1 Clone NC1 and Tc Clone L2 to iLRPPeptides Stimulant Th1 Clone NC1 Tc Clone L2 Medium .007, .008 .008,.009 P32 (aa 285-295) .015, .017 .016, .02  Intact iLRP  .24, .27   .93,.91  P1 (161-172) .016, .015 .015, .019 P2 (165-176) .017, .014  .02,.018 P3 (169-180) .016, .014 .017, .014 P4 (173-184) .015, .017 .018,.02  P5 (177-188) .017, .016 .014, .017 P6 (181-192) .015, .018 .015,.016 P7 (185-196) .014, .017 .016, .02  P8 (189-200) .015, .016 .014,.02  P9 (193-204) .016, .018 .017, .019 P10 (197-208) .014, .018 .015,.017 P11 (201-212) .014, .017 .014, .016 P12 (205-216) .018, .015 .017,.015 P13 (209-220) .017, .015 .017, .02  P14 (213-224) .015, .017 .014,.016 P15 (217-228) .014, .016 .015, .017 P16 (221-232) .016, .015 .017,.02  P16 (221-232) .016, .015 .017, .02  P17 (225-236)  .16, .17   .96,.94  P18 (229-240)  .25, .28   .95, .97  P19 (233-244) .015, .017 .016,.018

TABLE 11a (SEQ ID NOS 45 & 46, respectively, in order of appearance)iLRP Peptide Sequences Clones NC1 and L2 Recognize Peptide Amino AcidRange Sequence P17 aa 225-236 EEFQGEWTAPAP P18 aa 229-240 GEWTAPAPEFTA

Class II MHC-bound peptides tend to vary in length between 8 and 30amino acids. Most class II MHC-bound peptide epitopes are 9-11 aminoacids in length; the length of the epitope recognized by Th1 clone NC1was deduced to be 10 amino acids. Because the clone responded best topeptide 18, but significantly to peptide 17, it was deduced that cloneNC1 recognized the epitope GEWTAPAPEF(SEQ ID NO: 16). Peptide 17 has allof that, but the last two amino acids E and F. The proliferation to eventhese peptides as well as to intact iLRP was lower than what was usuallyobserved with Th cells. But the NC1 clone was derived from a mouse thathad never been immunized with iLRP, but just had this clone present inits spleen. Therefore, it was a naive T cell, not a memory T cell, soits T cell antigen receptor had a lower affinity reaction with the iLRPpeptide it recognized. Since CD8 Tc clone L2 proliferated identically topeptides 17 and 18, the deduced epitope for the L2 Tc clone was thecommon 8 amino acid sequence to both of those peptides, namelyGEWTAPAP(SEQ ID NO: 8).

Analysis of Specificity of iLRP-Specific Th1 Cell Clones Reactive toiLRP Peptide Spanning Amino Acids 243-295

As before, proliferation assays using ELISA measurement of BudRincorporation to measure proliferation of T cells cultured with variousiLRP peptides and T cell-depleted, irradiated, syngeneic spleen cells asantigen presenting cells all in medium containing 100 U/ml of IL-2 weredone. The cloned cells were cultured with antigen-presenting cells and100 ng/well of an irrelevant iLRP peptide as a negative control, intactiLRP protein as a positive control, or overlapping 12-mer iLRP peptidesthat spanned the 243-295 region. After 24 hours, BUdR was added and theculture continued for another 24 hours, at which time, the cells wereharvested and assayed for BUdR incorporation as per the instructions ofBioTrak Cell Proliferation ELISA System. The data presented below inTable 12 are the A₄₅₀ readings on the wells after the assay wascomplete. Deduced epitopes are set forth in Table 13.

TABLE 12 Proliferation Results (A₄₅₀) of Th1 Clones to iLRP Peptides Th1Clones Stimulant NC4 M2 H1 L3 Medium .005, .008 .007, .008 .007, .005.008, .009 P1 (161- .017, .015 .017, .018 .015, .02   .02, .015 172)Intact iLRP  .23, .27   .87, .91   .95, .96   .95, .98  P19 (233- .017,.014 .017, .02  .018, .02  .019, .015 244) P20 (237- .015, .016 .015,.019 .019, .018  .85, .83  248) P21 (241- .015, .016 .018, .017  .02,.02   .96, .98  252) P22 (245- .015, .017 .017, .016 .019, .017 .018,.02  256) P23 (249- .018, .014  .81, .83  .017, .016  .02, .016 260) P24(253- .017, .015  .93, .95  .016, .02  .017, .016 264) P25 (257- .018,.014 .018, .02  .016, .02  .017, .02  268) P26 (261- .015, .017  .02,.015  .02, .015 .016, .019 272) P27 (265- .017, .014 .015, .018 .017,.015 .015, .018 276) P28 (269- .015, .016 .019, .02  .016, .017 .016,.018 280) P29 (273- .014, .016  .02, .017  .85, .83  .014, .017 284) P30(277- .015, .018 .016, .017  .96, .98  .014, .02  288) P31 (281-  .17,.15  .016, .018 .015, .019  .02, .02  292) P32 (285-  .28, .26  .016,.02   .02, .017 .017, .018 295)

TABLE 13 (SEQ ID NOS 47-54, respectively, in order of appearance) iLRPPeptide Sequences Clones NC4, M2, H1, and L3 Recognize Peptide AminoAcid Range Sequence P20 aa 237-248 EFTAAQPEVADW P21 aa 241-252AQPEVADWSEGV P23 aa 249-260 SEGVQVPSVPIQ P24 aa 253-264 QVPSVPIQQFPT P29aa 273-284 TEDWSAAPTAQA P30 aa 277-288 SAAPTAQATEWV P31 aa 281-292TAQATEWVGATT P32 aa 285-295 TEWVGATTDWS

Since the clone NC4 responded best to peptide 32, but significantly topeptide 31, it was deduced that clone NC4 recognized the epitopeTEWVGATTDW(SEQ ID NO: 20) (amino acids 285-294). Peptide 31 has all ofthat except the last two amino acids D and W. Like the proliferation ofNC1 to intact iLRP or appropriate iLRP peptides, the proliferation ofNC4 to these peptides as well as to intact iLRP was lower than what wasobserved with the other Th cells assayed. This is because both NC1 andNC4 were clones derived from normal mice and therefore, not memory Tlymphocytes and so had lower affinity binding of iLRP. Because clone M2responded best to peptide 24, but significantly to peptide 23, it wasdeduced that clone M2 recognized the epitope QVPSVPIQQF(SEQ ID NO: 18)(amino acids 253-262). Peptide 23 has all of that except the last twoamino acids Q and F. Since clone H1 responded best to peptide 30, butsignificantly to peptide 29, it was deduced that clone H1 recognized theepitope SAAPTAQATE(SEQ ID NO: 19) (amino acids 277-286). Peptide 29 hasall of that except the last two amino acids T and E. Since clone L3responded best to peptide 21, but significantly to peptide 20, it wasdeduced that clone L3 recognized the epitope AQPEVADWSE(SEQ ID NO: 17)(amino acids 241-250). Peptide 20 has all of that except the last twoamino acids S and E.

Confirmation of Peptide Epitope binding to H-2^(d) Class I Proteins

Using a computer program developed by the University of Tuebingen,available through the internet for identification of potential bindingepitopes based on the particular MHC motifs, the amino acid sequence ofthe OFA/iLRP 30mer peptide that contains amino acids 136-166 was checkedfor L^(d)-bound motifs. See Table 14. Of the epitopes for the Ts and Tcclones which have been shown to be reactive with this region, theresults of the present analysis show that the same epitopes would bereactive with the L^(d) class 1 molecule.

TABLE 14 Reactive T cell clones & H2-Ld Anchor motif (SEQ ID NOS 39-44,respectively, in order of appearance) 31- aa136-147 EASYVNLPTIAL Ts 131- aa140-151     VNLPTIALCNTD Ts 2 31- aa144-155         TIALCNTDSPLRTc 3 31- aa148-159             CNTDSPLRYVDI Tc & 4 TH1 31- aa152-163                SPLRYVDIAIPC Tc & 5 TH1 31- aa156-167                    YVDIAIPCNNKG Tc 6 Binding Score (SEQ ID NO: 39) 31-aa136-147 E A SYVNLP T IAL  2 Ts 1 EASYVNLPTI AL 17 Ts EAS Y VNLPTI A L 1 Ts EASY V NLPTIA L 12 Ts (SEQ ID NO: 40) 31- aa140-151 V N LPTIAL CNTD  2 Ts 2 VN L PTIALC N TD  2 Ts VNL P TIALCN T D 15 Ts VNLP T IALCNTD  1 Ts (SEQ ID NO: 41) 31- aa144-155 T I ALCNTD S PLR  3 Tc 3 TI ALCNTDS P LR  1 Tc TIA L CNTDSP L R 10 Tc TIAL C NTDSPL R  1 Tc (SEQ IDNO: 42) 31- aa148-159 C N TDSPLR Y VDI  2 Tc & 4 TH1 CN T DSPLRY V DI  2Tc & TH1 CNT D SPLRYV D I  5 Tc & TH1 CNTD S PLRYVD I 17 Tc & TH1 (SEQID NO: 43) 31- aa152-163 S P LRYVDI A IPC 11 Tc & 5 TH1 SP L RYVDIA I PC 6 Tc & TH1 SPL R YVDIAI P C  2 Tc & TH1 (SEQ ID NO: 44) 31- aa156-167 YV DIAIPC N NKG  2 Tc 6 YV D IAIPCN N KG  3 Tc YVD I AIPCNN K G  3 TcYVDI A IPCNNK G  2 Tc

Using the same methodology, two additional OFA epitopes thatspecifically stimulate Tc cells were identified, mainly OFA (58-66)(e.g., LLLAARAIV) (SEQ ID NO: 55) and OFA (60-68) (e.g., LAARAIVAI) (SEQID NO: 56).

In Table 14 (as well as in Table 15 below), binding score refers to oneside of the epitope (agretope) to the MHC protein on theantigen-presenting cell. Since the T cell recognizes the other side ofthe sequence (epitope) in association with the MHC, high binding to theMHC indicates a greater likelihood of recognition of the epitope by theT cell. In these tables, the higher the binding score, the better thebinding. Thus, The amino acid residues set forth in bold are importantfor binding of the agretope to the MHC. As per the disclosure above,embodiments of the present invention include combinations of 2 or moreof the peptide sequences TIALCNTDS(SEQ ID NO: 11), TDSPLRYVD(SEQ ID NO:12), PLRYVDIAIP(SEQ ID NO: 57) and PLRYVDIAIP(SEQ ID NO: 57), that whenadministered to a human, will stimulate Tc cells (and in the case of thepeptides TDSPLRYVD(SEQ ID NO: 12) and PLRYVDIAIP(SEQ ID NO: 57),stimulate Th cells). Such combinations include 2 or more individualpeptides linked together e.g., via a spacer comprised of amino acids, orlinked to a common carrier.

The same 30mer sequence was analyzed in the same manner to identifypeptides that would be bound by human HLA class I protein A-2 (genotypeHLA-A*0201) as determined by the peptide motif required for binding bythe HLA-A2 protein. This produced 24 9mer peptides that should be boundby HLA-A2 class I protein and so presented as epitopes to T cells. SeeTable 15. This means that, although the peptides that will serve asepitopes for mouse T cells of a given strain will not necessarily be theexact epitopes for either another MHC-disparate strain of mouse, or forhumans, the same regions of the OFA/iLRP which are reactive with murineT cells will probably also be able to serve as a source of peptideepitopes of a slightly different sequence which will be recognized byhuman T cells. The peptides will be different for different MHChaplotypes to some extent and thus the exact epitopes recognized by Tcells will be slightly different, but some epitopes may be seen by Tcells of different individuals or species due to the high degree ofOFA/iLRP amino acid sequence conservation.

TABLE 15 Anchor Motif for human HLA-A*0201 within aa136–aa166 peptide(SEQ ID NOS 58-82, respectively, in order of appearance)aa136--------------------------------------------------------------aa166 EASYVNLPTIALCNTDSPLRYVDIAIPCNNKG Binding Score EASYVNLPT 5 ASYVNLPTI 18   SYVNLPTIA 7    YVNLPTIAL 21     VNLPTIALC 11     NLPTIALCN 11       LPTIALCNT 9        PTIALCNTD 7         TIALCNTDS10          IALCNTDSP 12           ALCNTDSPL 22            LCNTDSPLR 4            CNTDSPLRY 1              NTDSPLRYV 19              TDSPLRYVD 4                DSPLRYVDI 9                SPLRYVDIA 13                  PLRYVDIAI 15                  LRYVDIAIP 9                    RYVDIAIPC 1                    YVDIAIPCN 11                      VDIAIPCNN 3                      DIAIPCNNK 11                        IAIPCNNKG 13

Determination of OFA/iLRP Eptitopes for use in Humans

First, take 4 heparinized tubes of blood from the patient and send oneto the HLA typing laboratory to determine the HLA genotype of thepatient. The 3 other tubes of heparinized blood are used as a source ofT lymphocytes and antigen-presenting cells. The heparinized blood of thepatient is pooled and the peripheral blood mononuclear leucocytes (PBML)is purified by density gradient centrifugation in Ficoll-Paque Plus(Pharmacia Biotech, Piscataway, N.J.) using a modification of the methodof Boyum (Boyum, Scand. J. Clin. Lab. Invest. 21:97:S77 (1968)) aspreviously published and Rohrer et al., J. Immunol. 162:6880 (1999). Thepurified PBML is resuspended in RPMI-1640 medium and washed bycentrifugation 3 times and then a viability count done using Trypan Bluedye exclusion.

Second, the counted PBML is diluted to 5×10⁶ viable cells/ml inRPMI-1640 medium and then the cell suspension is split into twoaliquots. (a) One aliquot of cells serves as the source ofantigen-presenting cells in the proliferation assay. Deplete thisaliquot of T cells by negative selection on anti-CD3 monoclonal antibodycoated Petri plates using the method described in Wysocki et al., Proc.Natl. Acad. Sci. (USA) 75:2844 (1978), except that anti-CD3 antibody isused and that the anti-CD3 antibody is added and binds to the plates onthe day of the cell separation. See Boyum, Scand. J. Clin. Lab. Invest.21:97:S77 (1968). After incubation and removal of cells not adhering toanti-CD3-coated plates, the non-adherent cells (non-T cells) are washedby centrifugation in RPMI-1640 medium by centrifugation and X-irradiatedat 3000 R to inhibit their ability to proliferate. After X-irradiation,they are counted for viability using Trypan Blue dye exclusion and kepton ice until the proliferation assay is done. (b) The aliquot of cellsnot used for CD3⁺ cell (T cell) depletion is split in half andpositively selected for CD4 T cells and CD8 T cells using magnetic cellsorting. One half of the cells are incubated on ice with magnetic beadsthat are conjugated with anti-human CD4 monoclonal antibody. The otheraliquot is incubated with magnetic beads that are conjugated withanti-human CD8 monoclonal antibody. The incubations are done on ice for45 minutes. One tube (the anti-CD4 tube) is put in the field of aBecton-Dickinson Imag magnet, and the cells to which the antibody-coatedmagnetic beads have bound will bind to the side of the side of the tubehaving the magnet. The supernatant containing the cells not bearing themarker to which the antibody binds is removed by pipetting. New mediumis carefully added to the tube and the supernatant removed again. Afterthe non-bound cells are removed, the magnet will be removed and theanti-CD4 antibody-conjugated magnetic bead-bound cells are released fromthe side of the tube. Those CD4 T cells are removed by pipetting, washedby centrifugation in medium, and counted for viability by Trypan Bluedye exclusion. The same separation procedure is done to obtain CD8 Tcells using anti-human CD8 monoclonal antibody-conjugated magneticbeads. The two populations of T cells are diluted to 5×10⁶ viablecells/ml.

Third, the CD4 T cells and the CD8 T cells are assayed for proliferationto intact OFA/iLRP or 12-mer peptides that overlap by 4 amino acids, butspan the entire 295 amino acid sequence of OFA/iLRP (as was done toobtain the mouse epitope data presented in the examples above). Themethod described in Rohrer et al., J. Immunol. 162:6880 (1999) is usedto assess proliferation. Ninety-six (96) well plates are used and 0.1 mlof irradiated T cell-depleted PBML (obtained using the method describedin (a) above) are added to each well. That volume also contains 100 ngof intact OFA/iLRP, or a 12-mer peptide of OFA/iLRP. We then add 0.1 mlof CD4 or CD8 T cells from the cancer patient's peripheral blood(purifed by the method described in the first paragraph of the example,and in (b) above to all wells. We use 5×10⁴-5×10⁵ viable T cells/well,and incubate the plates containing the CD4 or CD8 T cells, irradiatedantigen-presenting cells plus intact OFA/iLRP or OFA/iLRP 12-merpeptides for 48 hours at 37° C. in a humidified 95% air/5% CO₂atmosphere. Twenty-four hours before harvest, 20 μl of5-bromodeoxyuridine (BUdR) are added to yield a total concentration of10 μM BUdR/well. The cell cultures are then returned to 37° C. in ahumidified 95% air/5% CO₂ atmosphere for the remaining 24 hours.Proliferation will be assayed using the Biotrak BUdR incorporation assay(Amersham, Arlington Heights, Ill.) as described previously. Afterpreparation and fixation of the labelled cells, precipitation of thecell DNA and enzyme-conjugated anti-BUdR antibody binding to the DNA,followed by washing and addition of a substrate for the enzyme that willproduce a colored product, plates are analyzed by a microELISA readerand 450 nm absorbance measured. By analysis of the absorbance valuescompared to negative controls, we determine which OFA/iLRP peptidesinduced proliferation by the patient's CD4 and CD8 T cells specific forOFA/iLRP. These data allow determination of the OFA/iLRP epitopesrecognized by the patient's T cells.

Fourth, because the cytotoxic T (Tc) cells and the IL-10-secreting,suppressor T (Ts) are both CD8 T cells and because induction of the Tscells inhibits Tc cell killing of tumor cells (Rohrer, et al., J.Immunol. 155:5719 (1995)), it is determined which of the epitopes thatinduce proliferation of the cancer patient's CD8 T cells are inducing Tcand which are inducing Ts cell activation. The Tc cells secreteinterferon-γ, but not IL-10 while the Ts cells secrete IL-10, but notinterferon-γ. We set up some of the CD8 and CD4 T cell cultures asdescribed above, in ELISPOT plates coated with either anti-interferon-γor anti-IL-10 antibody. The mixture of irradiated, autologousantigen-presenting cells, CD4 or CD8 T cells, and intact OFA/iLRP or thesame 12-mer OFA/iLRP peptides as in the proliferation assay are used.The cells are incubated at 37° C. in a humidified 95% air/5% CO₂atmosphere for 24-48 hours. At the end of that time, the cells arewashed off the wells and the biotinylated antibody (anti-interferon-γ oranti-IL-10) is added, incubated for 12 hours at 4° C., and then eachwell is washed to remove unbound antibody. This is followed by a 2-hourincubation with either streptavidin-alkaline phosphatase or horseradishperoxidase at room temperature. The appropriate substrate for the enzymeon the streptavidin is added, incubated at room temperature for 5-30minutes, the reaction stopped and the spots resulting from cytokinesecretion and being bound to the membrane bottom of the well are countedusing a Becton-Dickinson Immunospot Analyzer. By combining theproliferation assay data with the ELISPOT data, the OFA/iLRP peptidesinduce CD8 Tc cells (interferon-γ-secreting) and the peptides thatinduce CD8 Ts cells (IL-10-secreting) are identified. The same analysisof CD4 T cells allows a determination of whether different class IIHLA-presented peptides induce CD4 Th1 cells rather than CD4 Th2 cells.If they do, cell-mediated immunity against the tumor is augmented byimmunizing only with Th1-inducing OFA/iLRP peptides.

Once these data are obtained, resort is made to the database of HLAanchor motifs to determine which class I HLA protein is responsible forpresenting the peptides that are desired to be used. At this point, theHLA genotype of the patient and sequence of the OFA/iLRP peptides thatinduce T cell proliferation and interferon-γ-secretion are known. Afterthis analysis is done on enough patients, a large enough bank of datatelling which peptides need to be used for immunization in a patientwith a given HLA haplotype is accumulated. Thus, for a given HLAhaplotype, there is a given set of OFA/iLRP peptides that induceseffective immunotherapy of the tumor.

INDUSTRIAL APPLICABILITY

The present invention has applicability in cancer medicine and research.

All publications cited in the specification (e.g., the list of citationsbelow) are indicative of the level of skill of those skilled in the artto which this invention pertains. All these publications are hereinincorporated by reference to the same extent as if each individualpublication were specifically and individually indicated to beincorporated by reference. In addition, Applicants' provisional patentapplication No. 60/400,851, filed Aug. 2, 2002, is incorporated hereinby reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

LIST OF CITATIONS

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1. A composition comprising a plurality of oncofetal antigen (OFA)epitopes that specifically stimulate T cytotoxic lymphocytes in amammal, and a carrier, wherein said composition does not comprise anyOFA epitope that specifically stimulates T suppressor cells.
 2. Thecomposition of claim 1, wherein said carrier is an adjuvant.
 3. Thecomposition of claim 1, further comprising an adjuvant.
 4. Thecomposition of claim 1, wherein said carrier comprises a vesicle.
 5. Thecomposition of claim 4, wherein said vesicle comprises a liposome. 6.The composition of claim 1, wherein each of said epitopes is attached toa lipophilic group.
 7. The composition of claim 1, further comprising atleast one OFA epitope that specifically stimulates T helper lymphocytes.8. The composition of claim 7, wherein said at least one OFA epitopethat specifically stimulates T helper lymphocytes is attached to alipophilic group.
 9. The composition of claim 7, comprising at least twoOFA epitopes that specifically stimulate T helper lymphocytes.
 10. Thecomposition of claim 7, wherein said at least one OFA epitope thatspecifically stimulates T helper lymphocytes has about 8 to about 30amino acids.
 11. The composition of claim 10, wherein said at least oneOFA epitope that specifically stimulates T helper lymphocytes has about8 to about 12 amino acids.
 12. A method for preparing animmunotherapeutic composition for use in a human, comprising: (a)identifying a plurality of oncofetal antigen (OFA) epitopes thatspecifically stimulate T cytotoxic lymphocytes in the human; and (b)formulating two or more of the epitopes identified in (a) with acarrier, thus forming the immunotherapeutic composition.
 13. The methodof claim 12, further comprising c) identifying a plurality of oncofetalantigen (OFA) epitopes that specifically stimulate T helper lymphocytesin the human, and wherein b) comprises formulating one or more of theOFA epitopes identified in c) with the two or more epitopes identifiedin a) and the carrier.
 14. The composition of claim 1, wherein saidplurality of OFA epitopes stimulates different clones of T cytotoxiclymphocytes.
 15. The composition of claim 1, wherein each of saidplurality of OFA epitopes is present in said composition as a mixture.16. The composition of claim 1, wherein said plurality of OFA epitopesare linked together, thus forming one peptide.
 17. The composition ofclaim 1, wherein said plurality of OFA epitopes is linked to a commoncore structure.
 18. The composition of claim 17, wherein said commoncore structure is a multi-branched lysine or arginine core.
 19. Thecomposition of claim 1, wherein each of said plurality of OFA epitopesthat specifically stimulate T cytotoxic lymphocytes comprises about 8-12amino acid residues.
 20. The composition of claim 1, wherein saidplurality of OFA epitopes that specifically stimulate T cytotoxiclymphocytes are selected from the group consisting of RTWEKLLL (SEQ IDNO:6), NTGQRAVL (SEQ ID NO:7), CNTDSPLR (SEQ ID NO:9), YVDIAIPC (SEQ IDNO:10), and GEWTAPAP (SEQ ID NO:8).
 21. The composition of claim 7,wherein each of said OFA epitopes that specifically stimulate T helperlymphocytes is selected from the group consisting of SPLRYVDIAI (SEQ IDNO:15), GEWTAPAPEF (SEQ ID NO:16), AQPEVADWSE (SEQ ID NO:17), QVPSVPIQQF(SEQ ID NO:18), SAAPTAQATE (SEQ ID NO:19), and TEWVGATTDW (SEQ IDNO:20).
 22. An oncofetal antigen (OFA) fragment comprising an epitopethat specifically stimulates T helper lymphocytes in a mammal, whereinsaid composition does not comprise an OFA epitope that specificallystimulates T suppressor cells.
 23. A method of treating cancer in amammal, comprising administering to a mammalian cancer patient thecomposition of claim
 1. 24. The method of claim 23, wherein saidmammalian cancer patient is a human.